Re: human milk oligosaccharides 발효로 모유의 당분 얻기 방법 2024 NATURE

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Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

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• Open access
• Published: 13 June 2024

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

• Collin R. Barnum, 
• Bruna Paviani, 
• Garret Couture, 
• Chad Masarweh, 
• Ye Chen, 
• Yu-Ping Huang, 
• Kasey Markel, 
• David A. Mills, 
• Carlito B. Lebrilla, 
• Daniela Barile, 
• Minliang Yang & 
• Patrick M. Shih 

Nature Food volume 5, pages480–490 (2024)Cite this article

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Abstract

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

요약

모유 올리고당(HMO)은

유아의 건강과 발달을 지원하는 다양한 종류의 탄수화물입니다.

HMO의 막대한 건강상의 이점으로 인해

미생물 생산의 상업적 목표가 되었습니다.

그러나

약 200가지의 구조적으로 다양한 HMO를 대규모로 생산하는 것은

어려운 것으로 판명되었습니다.

여기에서는

식물의 탄수화물 동화 작용을 활용하여

다양한 HMO를 생산합니다.

이러한 다양성에는 락토-N-푸코펜타오스 I과 같은 고가 및 복합 HMO가 포함됩니다.

유전자 변형 식물에서 생산된 HMO는

강력한 비피더스균 생성 특성을 제공하여

성인 및 유아 건강에 잠재적으로 적용될 수 있는

프리바이오틱 보충제로서의 역할을 할 수 있는 능력을 나타냅니다.

기술경제학적 분석에 따르면, 식물에서 HMO를 생산하는 것은 미생물 생산 플랫폼보다 저렴한 가격으로 특정 HMO를 대규모로 생산할 수 있는 방법을 제공합니다. 우리의 작업은 식물을 활용하여 저비용으로 지속 가능한 HMO 생산을 가능하게 한다는 가능성을 보여줍니다.

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Main

Human milk is a complete and comprehensive food evolved to nourish and protect infants. A key component to the distinct bioactive properties of human milk is the presence of a wide diversity of human milk oligosaccharides (HMOs) which are well documented in establishing the nascent gut microbiota of infants to prevent diseases and ensure healthy development1,2,3,4. While 75% of infants are supplemented with or exclusively fed infant formula in the first 6 months of life, current infant formulas are either devoid of HMOs or only contain one to two of the ~200 HMOs found in human milk, limiting the health outcomes of formula-fed infants3,5,6. In addition to their use for infant health, HMOs are being studied for their beneficial roles in adult health as a prebiotic to improve intestinal barrier function, lower gastrointestinal inflammation and treat irritable bowel diseases7,8,9,10,11; however, the study of HMO benefits in adults has been limited to a small subset of HMOs. Currently, commercial HMO production relies on microbial fermentation but, to date, microbial fermentation is only able to commercially produce two to five simple HMOs of the ~200 HMOs found in human milk at a scale suitable to supplement food products6,12,13. While five simple HMOs constitute a large portion of HMO mass in human milk, diverse HMOs with a range of linkages and degrees of polymerization enable the growth of beneficial gut microbes which have preferences for specific HMOs14,15. Thus, there is a need to develop biological platforms to produce a wider diversity of HMOs found in human milk, enabling the supplementation of food products for both infants and adults.

The combinatorial nature of glycosidic linkages, nucleotide sugar donors and oligosaccharide acceptor molecules enables the large diversity of HMOs found in human milk16. HMOs are composed of five distinct sugars—d-glucose (Glc), d-galactose (Gal), N-acetylglucosamine (GlcNAc), l-fucose (Fuc) and N-acetylneuraminic acid (Neu5Ac)—connected through various glycosidic linkages to generate a diverse range of molecular structures (Fig. 1a). HMO biosynthesis begins with the production of lactose which can be decorated with fucose or Neu5Ac to form a variety of trisaccharides and tetrasaccharides. Lactose can also be extended by glycosyltransferases through the addition of a GlcNAc-ß-1,3 or GlcNAc-ß-1,4 to form unbranched HMOs. Unbranched HMOs can be further extended by glycosyltransferases through the addition of a GlcNAc-ß-1,6 to produce branched HMOs (Fig. 1b). GlcNAc can undergo subsequent addition of Gal-ß-1,3 or Gal-ß-1,4 to form type I and type II HMOs, respectively (Fig. 1b). While HMOs can consist of all five monosaccharides, they are generally classified in three broad HMO groups on the basis of their composition: (1) neutral HMOs contain Glc, Gal and GlcNAc, (2) fucosylated HMOs contain a neutral core with one or more Fuc additions and (3) acidic HMOs contain a neutral core with one or more additions of Neu5Ac. Owing to the need for high amounts of nucleotide sugars and glycosylation potential in HMO biosynthesis, a suitable host must have robust sugar metabolism capable of managing the metabolic burden of HMO production.

메인

모유는

유아에게 영양을 공급하고 보호하기 위해 진화한 완전하고 포괄적인 식품입니다.

모유의 독특한 생체 활성 특성의 핵심 요소는

유아의 초기 장내 미생물 군집을 형성하여 질병을 예방하고 건강한 발달을 보장하는 것으로 잘 알려진

다양한 모유 올리고당(HMO)의 존재입니다1,2,3,4.

생후 6개월 동안 유아의 75%가 분유를 보충하거나 분유만 먹지만,

현재의 분유에는 HMO가 없거나 모유에 존재하는 200여 가지 HMO 중 1~2가지만 포함되어 있어

분유를 먹는 유아의 건강에 제한이 있습니다3,5,6.

유아 건강에 대한 사용 외에도,

장벽 기능을 개선하고,

위장 염증을 줄이며,

과민성 대장 질환을 치료하는 프리바이오틱으로서 성인 건강에 대한

HMO의 유익한 역할에 대한 연구가 진행되고 있습니다7,8,9,10,11;

요약 모유는 유아 성장에 있어 영양의 황금 표준이며, 그 영양적 가치는 주로 모유 올리고당(HMO)에 기인합니다. HMO는 유당과 지질 다음으로 모유에 가장 많이 함유된 성분으로, 유아의 소화 기관에서는 소화되지 않는 독특한 구조적 다양성을 가진 복합 당입니다. 프리바이오틱스 역할을 하는 HMO의 여러 가지 유익한 기능은 장내 미생물군과의 상호작용을 통해 직접 또는 간접적으로 발휘되는 것으로 여겨집니다. 예를 들어, 유익한 박테리아의 성장을 돕고, 병원성 미생물에 대한 저항력을 강화하며, 장 상피세포의 반응을 조절하는 등의 효과가 있습니다. 최근 연구에 따르면, HMO는 유아의 건강을 증진하고 질병의 위험을 줄일 수 있는 것으로 밝혀졌으며, 식품 첨가물 및 치료제로서의 잠재력을 보여주고 있습니다. 이 논문은 HMO가 유아 장내 미생물 군집에 미치는 영향에 관한 최근의 연구에 대해 논의하고, HMO의 유익한 효과의 기저에 있는 분자적 메커니즘에 중점을 두고 있습니다. 배경 모유 수유가 고대부터 아기를 먹이는 데 최적화된 진화적 수단일 뿐 아니라 유아 영양의 황금 표준이라는 것은 널리 알려진 사실이며, 세계보건기구(WHO)는 출생 후 첫 6개월 동안은 모유만 먹여야 한다고 규정하고 있습니다[1,2,3]. 모유 수유는 건강한 발달과 성장에 필요한 영양분을 아기에게 제공해 주는 한편, 위장 및 호흡기 감염으로부터 아기를 보호하고, 비만, 당뇨병, 아토피, 천식 등 다양한 질병의 발생률을 감소시킵니다 [6,7,8,9,10,11,12]. 또한, 모유 수유는 아기와 엄마 모두에게 유익합니다 [13]. 최근 구조적 특성화 분석 도구의 발전과 개발로 인해 과학자들은 풍부하고 다양한 모유 올리고당(HMO)이 특징인 모유의 구성을 확인하는 과정에 참여하고 있습니다 [14]. 19세기 말, 젖병 수유 유아의 생존율이 모유 수유 유아에 비해 훨씬 낮고 감염 가능성이 더 높다는 사실이 밝혀지면서, 유아기의 건강에 긍정적인 영향을 미치는 모유의 성분에 대한 과학적 관심이 높아졌습니다. 1900년, 모유 수유 유아와 비수유 유아의 대변에 있는 박테리아의 구성에 차이가 있다는 사실이 밝혀졌고, 비피더스균이 모유 수유 유아의 대변에 더 많이 존재하는 것으로 나타났습니다 [15]. HMO 연구에 50년 이상 더 노력한 결과, 모유의 비피더스균 생성 인자가 다당류와 N-아세틸글루코사민(GlcNAc)을 함유한 올리고당류(OS)로 확인되었습니다[16,17,18]. 현재 200개가 넘는 HMO가 확인되었으며, 장내 미생물과의 상호 작용을 통해 작용하는 것으로 여겨지는 HMO가 모유의 많은 유익한 효과를 설명하는 것으로 알려져 있습니다 [19]. 모유의 성분 중 락토스와 지질은 영아에게 가장 중요한 탄수화물 공급원이며, 평균 농도 30-70g/L의 탄수화물을 제공합니다 [20,21,22]. 락토스와 지질에 비해, HMO는 모유의 세 번째로 큰 고체 성분으로, 약간만 가수분해되어 결국 유아의 위장관에 축적됩니다. HMO는 복합 탄수화물이며, 유아의 소화 기관에 흡수되지 않는 특성 때문에 결장 내 특정 장내 미생물의 기질 역할을 하는 프리바이오틱스로 기능하는 것으로 알려져 있습니다 [23, 24]. 잘 조절되어 있지만, 매우 복잡하고 중요한 생물학적 과정인 신생아의 위장관에서 건강한 숙주-미생물 공생의 발달은 여전히 매우 취약한 시기입니다 [25, 26]. 모유를 먹는 유아의 장내 미생물 군집에는 비피더스균이 풍부하게 존재하는데, 이는 유아에게 안전하고 유익한 것으로 간주됩니다 [27]. 게다가, HMO는 병원성 박테리아, 바이러스, 원생 동물 기생충, 곰팡이 등 원인균에 대한 보호와 관련이 있는 것으로 점점 더 많이 알려져 있습니다. 이 리뷰는 HMO가 유아의 장내 미생물 군집에 미치는 영향에 관한 현재의 발전 상황과 HMO의 유익한 효과 및 그 뒤에 있는 메커니즘에 대한 중요한 통찰력을 제시합니다. HMO HMO의 화학 구조 HMO는 모유에 존재하는 다양한 OS 그룹을 지칭하는 총칭으로, 시알산(Sia) 또는 N-아세틸뉴라민산이라는 하나의 산성 단당류, 글루코사민(GlcNAc)으로 알려진 하나의 아미노당, l-푸코스(Fuc)라는 세 가지 단당류, d-갈락토스(Gal), d-글루코스(Glc) [28]. 이 다섯 가지 구성 요소의 조합은 다양하고 복잡하지만, 지금까지 약 200가지의 HMO만이 특성화되었고, 50가지의 HMO 조성은 모유에 존재하는 HMO의 99%를 차지하는 것으로 추정됩니다 [19, 24]. 모든 HMO는 환원 말단에 락토오스 코어(Galβ-1,4Glc)를 포함하고 있으며[29], 이는 β1-3 또는 β1-6 결합을 통해 Galβ1-3GlcNAc(락토-N-비오스, LNB, 1형 사슬) 또는 Galβ1-4GlcNAc(N-아세틸락토사민, 2형 사슬)에 효소적으로 더 길어질 수 있습니다. [30]. 게다가, 핵심 HMO 구조는 Sia를 통해 α2-3 또는 α2-6 결합 및/또는 Fuc를 통해 α1-2, α1-3 또는 α1-4 결합을 통해 말단 위치에 장식될 수 있습니다 [30]. 따라서, HMO는 크게 퓨코실화 OS(FucOS), 시알릴화 OS(SiaOS), 중성 OS(Fig. 1)의 세 가지 그룹으로 분류할 수 있습니다.

그러나 성인에 대한 HMO의 유익성에 대한 연구는 소수의 HMO에 국한되어 있습니다.

현재 상업용 HMO 생산은

미생물 발효에 의존하고 있지만,

현재까지 미생물 발효는 모유에 존재하는

약 200가지의 HMO 중 2~5가지의 단순한 HMO만 식품 보충에 적합한 규모로

상업적으로 생산할 수 있습니다6,12,13.

5가지의 단순한 HMO가

모유의 HMO 질량에서 큰 부분을 차지하지만,

다양한 HMO와 다양한 결합 및 중합도를 통해

특정 HMO를 선호하는 유익한 장내 미생물의 성장을 가능하게 합니다14,15.

따라서,

유아와 성인 모두를 위한 식품 보충을 가능하게 하는 모유에서 발견되는 HMO의 다양성을 넓힐 수 있는

생물학적 플랫폼을 개발할 필요가 있습니다.

글리코시드 결합, 뉴클레오티드 당 공여체, 올리고당 수용체 분자의 조합적 특성은

모유에 존재하는 HMO의 다양성을 가능하게 합니다16.

HMO는

d-글루코스(Glc), d-갈락토스(Gal), N-아세틸글루코사민(GlcNAc), l-푸코스(Fuc), N-아세틸뉴라민산(Neu5Ac) 등

5가지의 뚜렷한 당으로 구성되어 있으며,

다양한 글리코시드 결합을 통해 연결되어 다양한 분자 구조를 생성합니다(그림 1a).

HMO 생합성은

락토오스의 생산으로 시작되며,

락토오스는 푸코스 또는 뉴5아세틸로 장식되어

다양한 트리사카라이드와 테트라사카라이드를 형성할 수 있습니다.

락토오스는 또한 글리코실트랜스퍼라제에 의해

글루코노락토오스-베타-1,3 또는 글루코노락토오스-베타-1,4가 추가되어

비분지 HMO를 형성함으로써 확장될 수 있습니다.

분지되지 않은 HMO는

글리코실트랜스퍼라제를 통해 GlcNAc-ß-1,6을 추가하여 분지된 HMO를 생성함으로써

더욱 확장될 수 있습니다(그림 1b).

GlcNAc은 이후 Gal-ß-1,3 또

는 Gal-ß-1,4를 추가하여 각각 유형 I 및 유형 II HMO를 형성할 수 있습니다(그림 1b).

HMO는 5가지 단당류로 구성될 수 있지만,

일반적으로 그 구성에 따라 크게 세 가지 HMO 그룹으로 분류됩니다:

(1) 중성 HMO는 Glc, Gal, GlcNAc를 포함하고,

(2) 푸코실화 HMO는 1개 이상의 Fuc가 추가된 중성 코어를 포함하고,

(3) 산성 HMO는 1개 이상의 Neu5Ac가 추가된 중성 코어를 포함합니다.

HMO 생합성 과정에서 다량의 뉴클레오티드 당과 글리코실화 가능성이 필요하기 때문에,

적합한 숙주는 HMO 생산의 대사 부담을 관리할 수 있는 강력한 당 대사 능력을 가져야 합니다.

Fig. 1: Production of all three HMO classes in planta.

a, HMOs are composed of Glc, Gal, Fuc, GlcNAc and/or Neu5Ac connected via Gal-ß-1,3/4, GlcNAc-ß-1,3/6, Fuc-α-1,2/3/4 or Neu5Ac-α-2,3/6 glycosidic linkages. b, HMOs can be divided into branched, unbranched, type I and/or type II HMOs. c, HMO biosynthetic pathways used in this study for HMO production in planta. d, Extracted ion chromatograms (EIC) showing the identification of 2′FL, 3′SL, 6′SL, LNFPI, LSTa, LSTc and LNT/LNnT in extracts of individual plant leaves using LC–MS/MS (Q Exactive, Thermo Fisher Scientific). Red, blue and purple colouring denote fucosylated, neutral and acidic HMOs, respectively. Some further peaks are present due to in-source fragmentation of larger oligosaccharides with no available standards.

a, HMO는 Gal-ß-1,3/4, GlcNAc-ß-1,3/6, Fuc-α-1,2/3/4 또는 Neu5Ac-α-2,3/6 글리코시드 결합을 통해 연결된 Glc, Gal, Fuc, GlcNAc 및/또는 Neu5Ac으로 구성됩니다. b, HMO는 분지형, 비분지형, 유형 I 및/또는 유형 II HMO로 나눌 수 있습니다. c, 본 연구에서 식물체 내 HMO 생산에 사용된 HMO 생합성 경로. d, LC-MS/MS(Q Exactive, Thermo Fisher Scientific)를 사용하여 개별 식물 잎 추출물에서 2′FL, 3′SL, 6′SL, LNFPI, LSTa, LSTc 및 LNT/LNnT의 확인을 보여주는 추출 이온 크로마토그램(EIC). 빨간색, 파란색, 보라색은 각각 푸코실화, 중성, 산성 HMO를 나타냅니다. 더 큰 올리고당의 내부 단편화로 인해 표준 물질이 없는 일부 추가 피크가 존재합니다.

Unlike many microbes used in commercial fermentation, plants have evolved to create a wide range of glycans which encompass a diversity of nucleotide sugars from photosynthetically fixed CO2. As masters of sugar anabolism, plants are able to create vast amounts of complex oligosaccharides and polysaccharides17. This has led to commercial operations for the production of prebiotic oligosaccharides from plant biomass, such as ß-glucan, xylooligosaccharides, inulin or soy oligosaccharides18,19,20,21. Many of these products can either be purified or directly consumed as a food, providing an easy means of ingestion. Additionally, plants can be grown in open fields, requiring minimal inputs, limiting the need for expensive substrates and axenic conditions22. The robust sugar metabolism of plants and ability to be grown at agricultural scales make plants an ideal platform for the large-scale production of HMOs.

Owing to the unique advantages of plants as a platform for carbohydrate production, we tested their ability to produce a range of HMOs using both transient and stable expression‎ in Nicotiana benthamiana. Here, we report in planta production of neutral, fucosylated and acidic HMOs showcasing the intrinsic advantages of a plant-based production platform. Furthermore, we show that plant-produced HMOs provide selective growth of key bifidobacteria, indicating their potential prebiotic efficacy. Finally, we assess the economic viability of HMO production in planta compared to current microbial platforms.

상업적 발효에 사용되는 많은 미생물과 달리,

식물은 광합성을 통해 고정된 CO2로부터

다양한 뉴클레오티드 당을 포함하는 광범위한 당 사슬을 생성하도록 진화했습니다.

당의 동화 작용의 주인인 식물은

방대한 양의 복잡한 올리고당과 다당류를 생성할 수 있습니다17.

이 때문에 식물 바이오매스에서

베타글루칸, 자일로올리고당, 이눌린, 대두 올리고당18,19,20,21과 같은

프리바이오틱 올리고당을 생산하는 상업적 운영이 이루어지고 있습니다.

이러한 제품 중

많은 제품은 정제되거나 식품으로 직접 섭취될 수 있어 섭취가 용이합니다.

또한, 식물은 최소한의 투입물로 넓은 들판에서 재배할 수 있어 값비싼 기질과 무균 상태가 필요하지 않습니다22.

식물의 탄탄한 당 대사 능력과 농업 규모로 재배할 수 있는 능력 덕분에

식물은 HMO의 대규모 생산을 위한

이상적인 플랫폼이 되었습니다.

탄수화물 생산 플랫폼으로서의 식물의 독특한 장점 덕분에,

우리는 Nicotiana benthamiana에서 일시적 발현과 안정적 발현을 모두 사용하여

다양한 HMO를 생산하는 식물의 능력을 테스트했습니다.

여기에서는 식물 기반 생산 플랫폼의 고유한 장점을 보여주는

중성, 푸코실화, 산성 HMO의 식물 내 생산에 대해 보고합니다.

또한,

식물에서 생산된 HMO가

주요 비피더스균의 선택적 성장을 촉진하여 잠재적인 프리바이오틱 효능을 나타낸다는 것을 보여줍니다.

마지막으로,

현재의 미생물 플랫폼과 비교하여

식물 내 HMO 생산의 경제적 타당성을 평가합니다.

Results

Production of all three HMO classes in planta

Production of HMOs requires the expression‎ of glycosyltransferases capable of creating specific glycosidic linkages. While HMO biosynthesis in humans takes place in the Golgi by means of a largely unknown pathway23, various microbial enzymes are capable of generating HMOs in the cytosol. To produce HMOs in plants, we localized bacterial HMO biosynthetic enzymes to the cytosol for the production of neutral, fucosylated and acidic HMOs (Fig. 1c). We tested these pathways through transient expression‎ in N. benthamiana. Transient expression‎ permits relatively high throughput screening of biosynthetic pathways in planta by injecting strains of Agrobacterium tumefaciens into plant leaves, allowing the Agrobacterium to insert genes for HMO biosynthetic genes into the plant cell24. Leaves transiently expressing HMO biosynthetic pathways were subjected to liquid–liquid extraction, C18 solid-phase extraction (SPE) and porous graphitic carbon (PGC) SPE before characterization by mass spectrometry (MS) (Supplementary Fig. 1).

Neutral HMOs function as the core scaffolds of other more complex HMOs (fucosylated and acidic); thus, we first targeted the type I and type II neutral HMO core structures, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Expression‎ of a neutral HMO biosynthetic pathway using two ß-1,4-galactosyltransferases (GalTPM1141 (ref. 25), Hp0826 (ref. 26)), one ß-1,3-galactosyltransferase (Cvß3GalT27) and one ß-1,3-N-acetylglucosaminyltransferease (NmLgtA28) resulted in the production of lactose and various neutral HMOs with degrees of polymerization ranging from three to seven. Notably, N. benthamiana transiently expressing this pathway produced the tetrasaccharides LNT (m/z 708.2559) and LNnT (m/z 708.2559), which represent principal type I and type II HMOs in human milk, respectively29 (Fig. 1d and Supplementary Table 1). Additionally, we identified the production of larger neutral oligosaccharides with varying degrees of polymerization using tandem mass spectrometry (MS/MS) fragmentation to determine the number of hexose and HexNAc sugars (Supplementary Table 1). This included several neutral isomers of pentasaccharides and heptasaccharides (Supplementary Table 1). Our findings demonstrate that plants have the ability to produce previously inaccessible oligosaccharides with various degrees of polymerization which may expand HMO functional bioactivity.

Following the success of generating type I and type II neutral HMOs, we examined the ability of plants to decorate neutral HMOs with fucose, as fucosylated HMOs are the most abundant class of HMO in human milk15. We transiently expressed an α-1,2-fucosyltransferase (Te2FT30) alongside the neutral HMO biosynthetic pathway to produce the most abundant fucosylated HMOs in human milk: 2′-fucosyllactose (2′FL) (m/z 489.1819) and lacto-N-fucopentaose I (LNFPI) (m/z 854.3136) (Fig. 1d and Supplementary Table 1). Additionally, several fucosylated hexasaccharide isomers were identified by m/z and MS/MS fragmentation (Supplementary Table 1). While the structure of each isomer could not be determined, each is composed of four hexoses, one HexNAc and one deoxyhexose, indicating that either LNFPI can be further decorated with hexose sugars or pentasaccharide neutral HMOs can be decorated with additional fucose.

While neutral and fucosylated HMOs represent most HMOs in human milk, acidic HMOs constitute the last main class found in mammalian milks which provide unique bioactivities as a result of the presence of N-acetylneuraminic acid31. Plants do not natively produce the donor molecule for production of acidic HMOs, CMP-Neu5Ac. To produce acidic HMOs, we simultaneously expressed the neutral HMO biosynthetic pathway, sialyltransferases and a mammalian pathway for the production of CMP-Neu5Ac (Supplementary Fig. 2)32. Expression‎ of an α-2,6-sialyltransferase (St6; ref. 33) alongside the neutral HMO biosynthetic pathway produced the acidic trisaccharide, 6′-sialyllactose (6′SL) (m/z 634.2191), of type II acidic HMO, sialyllacto-N-neotetraose c (LSTc) (m/z 999.3505) (Fig. 1d and Supplementary Table 1). Expression‎ of an α-2,3-sialyltransferase (PmST3; ref. 34) with the neutral HMO biosynthetic pathway produced a myriad of acidic HMOs, such as the acidic trisaccharide, 3′-sialyllactose (3′SL) (m/z 634.2187) and the acidic pentasaccharide, LSTd (m/z 999.3510) (Fig. 1d and Supplementary Table. 1). In addition to making several LST isomers in vivo, six isomers of acidic hexasaccharides were identified using m/z and MS/MS fragmentation. Each isomer was composed of four hexoses, one HexNAc and one Neu5Ac (Supplementary Table 1). Together, these results show the ability of plants to produce all three classes of HMOs combinatorially or simultaneously (Supplementary Fig. 3), including type I and type II structures, marking the greatest diversity of HMOs made in a single heterologous organism.

결과

식물 재배로 HMO 클래스 3종 모두 생산

HMO 생성을 위해서는

특정한 글리코시드 결합을 생성할 수 있는 글리코실트랜스퍼라제의 발현이 필요합니다.

인간에서 HMO 생합성은

대부분 알려지지 않은 경로를 통해

골지체에서 이루어지지만23,

다양한 미생물 효소는 세포질에서 HMO를 생성할 수 있습니다.

식물에서 HMO를 생성하기 위해,

우리는 중성, 푸코실화, 산성 HMO 생성을 위해

세균성 HMO 생합성 효소를 세포질에 국한시켰습니다(그림 1c).

우리는 N. benthamiana에서 일시적 발현을 통해 이러한 경로를 테스트했습니다. 일시적 발현은 Agrobacterium tumefaciens 균주를 식물 잎에 주입하여 식물체 내 생합성 경로를 비교적 높은 처리량으로 스크리닝할 수 있게 해줍니다. 이때 Agrobacterium은 HMO 생합성 유전자를 식물 세포에 삽입할 수 있습니다24. HMO 생합성 경로를 일시적으로 표현하는 잎은 질량 분석법(MS)으로 특성화하기 전에 액체-액체 추출, C18 고체상 추출(SPE), 다공성 흑연 탄소(PGC) SPE를 거쳤습니다(그림 1 참조).

중성 HMO는 다른 더 복잡한 HMO(펩티드화 및 산성)의 핵심 골격으로 기능합니다. 따라서, 우리는 먼저 유형 I과 유형 II 중성 HMO 핵심 구조인 락토-N-테트라오스(LNT)와 락토-N-네오테트라오스(LNnT)를 대상으로 삼았습니다. 두 개의 β-1,4-갈락토실트랜스퍼라제(GalTPM1141 (ref. 25)를 사용하는 중성 HMO 생합성 경로의 표현, Hp0826(ref. 26), 1개의 ß-1,3-갈락토실트랜스퍼라제(Cvß3GalT27) 및 1개의 ß-1,3-N-아세틸글루코사미닐트랜스퍼라제(NmLgtA28)는 3~7의 중합도를 갖는 락토스와 다양한 중성 HMO를 생산하는 결과를 가져왔습니다. 특히, 이 경로를 일시적으로 발현하는 N. benthamiana는 각각 모유에서 주요 유형 I과 유형 II HMO를 나타내는 테트라사카라이드 LNT(m/z 708.2559)와 LNnT(m/z 708.2559)를 생성했습니다29 (그림 1d 및 부록 표 1). 또한, 헥소스와 헥스나크 당의 수를 결정하기 위해 탠덤 질량 분석법(MS/MS) 단편화를 사용하여 중합도가 다양한 더 큰 중성 올리고당의 생산을 확인했습니다(보충표 1). 여기에는 펜타당과 헵타당의 여러 중성 이성질체가 포함됩니다(보충표 1). 우리의 연구 결과는 식물이 이전에 접근할 수 없었던 다양한 중합도를 가진 올리고당을 생산할 수 있는 능력이 있다는 것을 보여줍니다. 이는 HMO의 기능적 생체 활성을 확장할 수 있습니다.

제1형과 제2형 중성 HMO 생성의 성공에 이어, 우리는 중성 HMO를 푸코스로 장식하는 식물의 능력을 조사했습니다. 푸코실화 HMO가 모유에서 가장 풍부한 HMO 종류이기 때문입니다15. 우리는 중성 HMO 생합성 경로와 함께 α-1,2-푸코실트랜스퍼라제(Te2FT30)를 일시적으로 발현하여 모유에서 가장 풍부한 푸코실화 HMO를 생산했습니다. 2'-푸코실락토오스(2'FL) (m/z 489.1819)와 락토-N-푸코펜타오스 I(LNFPI) (m/z 854.3136) (그림 1d 및 부록 표 1). 또한, m/z와 MS/MS 단편화에 의해 여러 개의 푸코실화 헥사사카라이드 이성질체가 확인되었습니다(보충표 1). 각 이성질체의 구조는 확인할 수 없었지만, 각각 네 개의 헥소오스, 하나의 헥스나크, 하나의 데옥시헥소오스로 구성되어 있다는 사실은 LNFPI가 헥소오스 당으로 추가 장식될 수 있거나, 5당 중성 HMO가 추가 푸코스로 장식될 수 있음을 나타냅니다.

중성 및 푸코실화 HMO가 모유에 존재하는 대부분의 HMO를 차지하는 반면, 산성 HMO는 포유류 우유에서 발견되는 마지막 주요 클래스로, N-아세틸뉴라민산의 존재로 인해 독특한 생체 활성을 제공합니다31. 식물은 산성 HMO, CMP-Neu5Ac를 생산하기 위한 기증 분자를 본래 생산하지 않습니다. 산성 HMO를 생산하기 위해, 중성 HMO 생합성 경로, 시알릴트랜스퍼라제, 그리고 포유류 CMP-Neu5Ac 생산 경로를 동시에 발현시켰습니다(그림 2 부록)32. α-2,6-시알릴트랜스퍼라제(St6; 참고 33)의 발현과 중성 HMO 생합성 경로를 함께 사용하면 산성 HMO 유형 II의 산성 트리사카라이드 6′-시알릴락토오스(6′SL)(m/z 634.2191)가 생성됩니다. sialyllacto-N-neotetraose c (LSTc) (m/z 999.3505) (그림 1d 및 부록 표 1). 중성 HMO 생합성 경로를 가진 α-2,3-sialyltransferase(PmST3; 참고 문헌 34)의 발현은 산성 트리사카라이드와 같은 무수한 산성 HMO를 생성했습니다. 3'-sialyllactose (3'SL) (m/z 634.2187)와 산성 오당류 LSTd (m/z 999.3510) (그림 1d 및 부록 표 1). 생체 내에서 여러 LST 이성질체를 생성하는 것 외에도, m/z와 MS/MS 단편화를 사용하여 산성 6당류의 6가지 이성질체가 확인되었습니다. 각 이성질체는 4개의 헥소오스, 1개의 HexNAc, 1개의 Neu5Ac로 구성되어 있습니다(보충표 1). 이 결과를 종합해 보면, 식물이 유형 I과 유형 II 구조를 포함하여 세 가지 종류의 HMO를 조합적으로 또는 동시에 생산할 수 있는 능력을 보여줍니다(보충그림 3). 이는 단일 이종 유기체에서 생산되는 HMO의 다양성이 가장 크다는 것을 의미합니다.

Optimized production of complex fucosylated HMOs

Microbial production platforms suffer from an inability to produce HMOs with higher degrees of polymerization at large scales, leaving many larger, more complex HMOs understudied. LNFPI is a fucosylated pentasaccharide that is the second most abundant fucosylated HMO. Despite its high abundance in breast milk, LNFPI has remained recalcitrant to fermentative production in microbes, limiting efforts to study its potential health benefits. Therefore, we sought to optimize the production of LNFPI in planta by overexpressing the requisite nucleotide sugar biosynthetic pathways. We transiently expressed the biosynthetic pathway for LNFPI (Fig. 2a) alongside pathways for the production of UDP-galactose, UDP-N-acetylglucosamine (UDP-GlcNAc) and GDP-fucose and quantified LNFPI production (Fig. 2b). Expression‎ of the LNFPI pathway with the GDP-fucose pathway increased production of LNFPI by 32.9% (1,075.03 μg g−1 of dry weight) compared to the expression‎ of the LNFPI pathway alone (808.91 μg g−1 of dry weight), indicating that GDP-fucose is limiting in N. benthamiana (Fig. 2c). Surprisingly, overexpression‎ of the GDP-fucose pathway also resulted in the production of lactodifucotetraose (LDFT) (m/z 635.2394) and lacto-N-difuco-hexaose I (LNDFHI) (m/z 1,000.3720) (Supplementary Table 1) despite not expressing an α-1,3- or α-1,4-fucosyltransferase, indicating the presence of native plant fucosyltransferases capable of glycosylating HMOs. Overexpression‎ of all other nucleotide sugar pathway combinations resulted in similar or lower levels of LNFPI production compared to expression‎ of the LNFPI pathway alone.

복잡한 fucosylated HMO의 최적화된 생산

미생물 생산 플랫폼은 고분자화 정도가 높은 HMO를 대규모로 생산할 수 없어, 더 크고 복잡한 HMO가 많이 연구되지 않은 채로 남아 있습니다. LNFPI는 두 번째로 풍부한 푸코실화 HMO인 푸코실화 펜타사카라이드입니다.

모유에 풍부하게 존재함에도 불구하고,

LNFPI는 미생물 발효 생산에 있어 난제로 남아 있어,

잠재적인 건강상의 이점을 연구하려는 노력을 제한하고 있습니다.

따라서, 우리는 필요한 뉴클레오티드 당 생합성 경로를 과발현함으로써 식물체 내 LNFPI 생산을 최적화하고자 했습니다. 우리는 UDP-갈락토오스, UDP-N-아세틸글루코사민(UDP-GlcNAc), GDP-푸코오스 생산 경로와 함께 LNFPI 생합성 경로(그림 2a)를 일시적으로 발현하고 LNFPI 생산량을 정량화했습니다(그림 2b). GDP-fucose 경로를 포함한 LNFPI 경로의 발현은 LNFPI 경로 단독 발현(건조 중량 808.91μgg-1)에 비해 LNFPI의 생산을 32.9% 증가시켰습니다(건조 중량 1,075.03μgg-1). 이는 GDP-fucose가 N. benthamiana에서 제한적임을 나타냅니다(그림 2c). 놀랍게도 GDP-fucose 경로의 과다 발현은 α-1,3- 또는 α-1,4-fucose를 발현하지 않음에도 불구하고 lactodifucotetraose(LDFT) (m/z 635.2394)와 lacto-N-difuco-hexaose I(LNDFHI) (m/z 1,000.3720)의 생산을 초래했습니다(보충표 1). HMOs를 글리코실화할 수 있는 토종 식물 푸코실트랜스퍼라제의 존재를 나타냅니다. 다른 모든 뉴클레오티드 당 경로의 과발현은 LNFPI 경로의 발현과 비교했을 때, 비슷한 수준 또는 더 낮은 수준의 LNFPI 생산을 초래했습니다.

Fig. 2: Manipulation of nucleotide sugar biosynthetic pathways modulates HMO profiles in planta.

a, HMO biosynthetic pathway expressed for the production of LNFPI. b, Nucleotide sugar biosynthetic pathways expressed with LNFPI pathway. c, Quantification of LNFPI production through expression‎ of LNFPI biosynthetic pathway alongside combinatorially expressed nucleotide sugar biosynthetic pathways using an internal calibration curve obtained with an Agilent 6530 Accurate-Mass Q-ToF MS. The middle bar represents the median. Upper and lower whiskers correspond to the largest and smallest values within 1.5 × the interquartile range, respectively. Upper and lower hinges represent the third and first quartiles, respectively. Statistical analysis was conducted using a heteroscedastic two-tailed Student’s t-test with the LNFPI pathway expressed alone used as the reference group. *P < 0.05. P values are: LNFPI + fucose, 0.030; LNFPI + GlcNAc, 0.012; LNFPI + fucose + GlcNAc, 0.01; LNFPI + GlcNAc + Gal, 0.01; LNFPI + fucose + GlcNAc + Gal, 0.043. A sample size of three leaves was used for each experiment. d, Effect of nucleotide sugar biosynthetic pathway overexpression‎ on HMO profile produced using the LNFPI pathway. Values reflect normalized peak area. Hexose, HexNAc, deoxyhexose (Deoxyhex) determined using m/z and MS/MS fragmentation. We performed mass spectral analysis on an Agilent Q-ToF MS.

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The overall profile of oligosaccharides produced was altered by overexpressing nucleotide sugar biosynthetic pathways (Fig. 2d). The number of hexose (Glc, Gal), HexNAc (GlcNAc) and deoxyhexose (Fuc) sugars in each oligosaccharide identified was determined through identification by means of m/z and MS/MS fragmentation and peak area was normalized using a LNFPI calibration curve. Overexpression‎ of the UDP-GlcNAc and LNFPI pathways increased the relative amount of neutral oligosaccharides containing a hexose and HexNAc compared to expression‎ of the LNFPI pathway alone (Fig. 2d). Overexpression‎ of the GDP-fucose and LNFPI pathways resulted in a shift in the overall oligosaccharide composition, favouring the production of oligosaccharides containing at least one deoxyhexose, indicating an increase in the level of fucosylated oligosaccharides (Fig. 2d). These results demonstrate that tailoring the availability of nucleotide sugars enables control over the ratio of HMOs produced.

Because scaling HMO production in plants requires the growth of stably transformed plants, we developed transgenic lines of N. benthamiana expressing the LNFPI biosynthetic pathway. We generated two constructs for the constitutive production of 2′FL and LNFPI in transgenic N. benthamiana (Fig. 3a). HMO10 contains genes that encode four enzymes required to produce lactose, 2′FL, LNTII, LNT and LNFPI connected by means of 2A peptides35 to allow several coding sequences to be driven by a single constitutive promoter. To explore the effects of overexpressing portions of the GDP-fucose pathway, we also generated stable lines expressing HMO11, which contains a GDP-d-mannose-4,6-dehydratase (Gmd25) from the GDP-fucose pathway. Gmd transiently expressed alongside the neutral HMO pathway altered the HMO profile of plants in a similar way to expression‎ of the full GDP-fucose pathway (Supplementary Fig. 4).

생성된 올리고당의 전체적인 프로필은 뉴클레오티드 당 생합성 경로를 과발현함으로써 변경되었습니다(그림 2d). 확인된 각 올리고당의 헥소당(Glc, Gal), 헥스-N-아세틸당(GlcNAc), 데옥시헥소당(Fuc) 당의 수는 m/z 및 MS/MS 단편화를 통해 확인되었고, 피크 면적은 LNFPI 보정 곡선을 사용하여 정규화되었습니다. UDP-GlcNAc와 LNFPI 경로의 과다 발현은 LNFPI 경로 단독 발현에 비해 6탄당과 HexNAc를 포함하는 중성 올리고당의 상대적 양을 증가시켰다(그림 2d). GDP-fucose와 LNFPI 경로의 과다 발현은 전체 올리고당 구성의 변화를 초래하여, 적어도 하나의 데옥시헥소스를 포함하는 올리고당의 생산을 촉진하여, fucosylated 올리고당의 수준이 증가함을 나타냅니다(그림 2d). 이러한 결과는 뉴클레오티드 당의 가용성을 조정함으로써 생산되는 HMO의 비율을 조절할 수 있음을 보여줍니다.

공장에서 HMO 생산을 확대하려면 안정적으로 변형된 공장의 성장이 필요하기 때문에, 우리는 LNFPI 생합성 경로를 발현하는 N. benthamiana의 형질전환 계통을 개발했습니다. 우리는 트랜스제닉 N. benthamiana에서 2′FL과 LNFPI의 구성적 생산을 위한 두 가지 구조를 생성했습니다(그림 3a). HMO10은 2A 펩티드를 통해 연결된 락토스, 2′FL, LNTII, LNT, LNFPI를 생산하는 데 필요한 네 가지 효소를 암호화하는 유전자를 포함하고 있어, 여러 개의 코딩 서열이 하나의 구성적 프로모터에 의해 구동될 수 있도록 합니다35. GDP-fucose 경로의 일부를 과발현했을 때의 효과를 알아보기 위해, 우리는 GDP-fucose 경로에서 GDP-d-mannose-4,6-dehydratase(Gmd25)를 포함하는 HMO11을 발현하는 안정된 계통을 생성했습니다. 중성 HMO 경로와 함께 일시적으로 발현된 GMD는 전체 GDP-퓨코스 경로의 발현과 유사한 방식으로 식물의 HMO 프로파일을 변경했습니다(그림 4).

Fig. 3: Production of HMOs in stably transformed plants.

a, Constructs used in creation of stable lines containing biosynthetic enzymes for the production of LNFPI. b, Photos of 4-week-old transgenic N. benthamiana. c, Concentration of LNFPI produced in leaves of each stable line. d, Concentration of 2′FL produced in leaves of each stable line. For quantification, three leaves from each plant were analysed separately. Quantification of LNFPI and 2′FL obtained with a Thermo Fisher Scientific Q Exactive Mass spectrometer. LB, left border; 2A , 2A peptide; RB, right border; Px, promoter; Tx, terminator; WT, wild type. The middle bar represents the median. Upper and lower whiskers correspond to the largest and smallest values within 1.5 × the interquartile range, respectively. Upper and lower hinges represent the third and first quartiles, respectively. A sample size of three leaves was used for each experiment.

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Transgenic T0 HMO-producing stable lines were assessed for fucosylated HMO yield. Most transgenic plants showed no drastic phenotypes compared to wild-type plants (Fig. 3b) and we conducted quantitative PCR with reverse transcription (RT–qPCR) analysis to confirm‎ the expression‎ transgenes (Supplementary Fig. 5). LNFPI and 2′FL were detected in leaves of transgenic N. benthamiana expressing both HMO10 and HMO11. Highest producing LNFPI lines accumulated an average concentration of 6.88 µg g−1 dry weight (Fig. 3c). Leaves from HMO11 no. 5 produced the highest concentration of 2′FL, reaching an average concentration of 130.35 µg g−1 dry weight (Fig. 3d). The low abundance of LNFPI in stable lines compared to transient expression‎ could indicate that the ß-1,3-N-acetylglucosaminyltransferase and ß-1,3-galactosyltransferase suffer from altered activity due to the presence of 2A peptides or lower expression‎. Together, these results show the ability to produce and optimize a diversity of HMOs from photosynthetically fixed CO2, laying the foundation for future efforts to create high-HMO-yielding transgenic plants for commercial HMO production.

Purification and functional characterization of HMOs from plants

Mixtures of prebiotic sugars can have varying effects on the enrichment of beneficial gut microbes36. Therefore, we sought to assess the bifidogenic activity of extracts from HMO-producing plants; however, crude plant extracts can contain chemicals that interfere with bacterial growth assays, such as simple sugars (glucose, fructose and sucrose) and antimicrobial specialized metabolites. Therefore, we developed a method to extract and purify HMOs from N. benthamiana transiently expressing the biosynthetic pathways for LNFPI (Fig. 2a) and GDP-fucose (Fig. 2b) using an optimized extraction and purification process. Briefly, we performed a water extraction, yeast fermentation to remove simple sugars and a two-step resin adsorption with polyvinylpolypyrrolidone (PVPP) and C18 SPE. This resulted in an HMO-rich extract that contained negligible amounts of simple sugars and phenolic compounds (Supplementary Table 3). The HMO extract contained target fucosylated HMOs (Supplementary Fig. 6), including LNFPI, 2′FL and LNDFHI (Supplementary Table 4). The extract also contained a variety of additional oligosaccharides without assigned structures, which were composed of combinations of hexose, deoxyhexose and HexNAc sugars (Supplementary Fig. 6). These represent potentially non-natural oligosaccharide structures which could provide potential health benefits. Overall, these results show the ability to isolate HMOs from plants, improving their promise as an HMO production platform.

To assess the bifidogenic activity of plant-produced HMOs, we conducted growth assays to compare the effects of plant-produced HMOs to HMOs derived from human milk. We chose to assess the effects of plant-derived HMOs on Bifidobacterium longum subsp. infantis ATCC 15697 (BLI 15697) as it is a known HMO consumer37. We also included Bifidobacterium animalis subsp. lactis ATCC 27536 (BLAC 27536) as a negative control which does not consume HMOs38 but will grow on simple sugars that could be present in plant extracts or human milk. BLI 15697 grown in media containing plant-derived HMOs showed increases in optical density OD600 nm similar to that of BLI 15697 in HMO isolated from human milk, demonstrating that plant-produced HMOs possess the same selective bifidogenic activity as HMOs isolated from human milk (Fig. 4b). As expected, BLAC 27536 showed no growth in either plant-derived HMOs or HMO isolated from human milk, indicating that both extracts contained a minimal amount of simple sugars present (Fig. 4b). Together, these results demonstrate the ability of purified, plant-produced HMOs to mimic the bifidogenic activity of HMOs produced in humans.

식물에서 추출한 HMO의 정제 및 기능적 특성 분석

프리바이오틱 당의 혼합물은

유익한 장내 미생물의 증식에 다양한 영향을 미칠 수 있습니다36.

따라서,

우리는 HMO를 생산하는 식물에서 추출한 추출물의 비피도생성 활성을 평가하고자 했습니다.

그러나,

원료 식물 추출물에는

단순 당(포도당, 과당, 자당)과 항균성 특수 대사 산물과 같은

박테리아 성장 분석을 방해하는 화학 물질이 포함되어 있을 수 있습니다.

따라서, 우리는 최적화된 추출 및 정제 과정을 사용하여 LNFPI(그림 2a)와 GDP-fucose(그림 2b)의 생합성 경로를 일시적으로 발현하는 N. benthamiana에서 HMO를 추출하고 정제하는 방법을 개발했습니다.

간단히 말해서,

우리는 물 추출, 단순 당을 제거하기 위한

효모 발효, 폴리비닐폴리피롤리돈(PVPP)과 C18 SPE를 이용한 2단계 수지 흡착을 수행했습니다.

그 결과,

HMO가 풍부하고 단순당과 페놀 화합물의 함량이 무시할 만한 수준인

추출물이 만들어졌습니다(보충표 3).

HMO 추출물에는 LNFPI, 2′FL, LNDFHI(보충표 4)를 포함한 표적 푸코실화 HMO(Supplementary Fig. 6)가 포함되어 있었습니다. 추출물에는 또한 할당된 구조가 없는 다양한 추가 올리고당이 포함되어 있었는데, 이들은 6탄당, 데옥시6탄당, 헥스-N-아세틸뉴라민산의 당의 조합으로 구성되어 있었습니다(그림 6 참조). 이것들은 잠재적인 건강상의 이점을 제공할 수 있는 비자연적인 올리고당 구조를 나타냅니다. 전반적으로, 이러한 결과는 식물에서 HMO를 분리할 수 있는 능력을 보여줌으로써 HMO 생산 플랫폼으로서의 가능성을 향상시킵니다.

식물에서 생산된 HMO의 비피더스균 생성 활성을 평가하기 위해, 식물에서 생산된 HMO와 모유에서 추출된 HMO의 효과를 비교하는 성장 분석을 실시했습니다. 식물에서 생산된 HMO가 Bifidobacterium longum subsp. infantis ATCC 15697(BLI 15697)에 미치는 영향을 평가하기로 결정했습니다. 이 균주는 HMO의 알려진 소비자이기 때문입니다37. 또한 Bifidobacterium animalis subsp. lactis ATCC 27536(BLAC 27536)을 음성 대조군으로 포함시켰습니다. 이 균주는 HMO를 섭취하지 38 않지만 식물 추출물이나 모유에 존재할 수 있는 단순 당류에서 자랄 수 있습니다. 식물 유래 HMO가 함유된 배지에서 성장한 BLI 15697은 인간 모유에서 분리된 HMO와 유사한 광학 밀도 OD600 nm의 증가를 보였으며, 이는 식물에서 생산된 HMO가 인간 모유에서 분리된 HMO와 동일한 선택적 비피더스균 생성 활성을 가지고 있음을 보여줍니다(그림 4b). 예상대로, BLAC 27536은 식물 유래 HMO나 모유에서 분리된 HMO 모두에서 성장하지 않았습니다. 이는 두 추출물 모두 최소량의 단당류를 함유하고 있음을 나타냅니다(그림 4b). 이러한 결과는 정제된 식물에서 생산된 HMO가 인간에서 생산된 HMO의 비피도생성 활성을 모방할 수 있음을 보여줍니다.

Fig. 4: Optimized purification protocol developed for functional analysis of plant-produced HMOs.

이 그림은 (a) 식물조직으로부터 기능성 물질(특히 탄수화물·페놀류 화합물 등)을 추출‧정제하고 발효 공정을 거친 뒤, 다양한 분석·특성평가를 수행하는 전반적 프로토콜과, (b) 그 최종 물질 또는 분획물이 특정 조건에서 시간에 따라 어떻게 반응(예: 미생물 성장, 광학 측정 등)하는지를 나타낸 그래프입니다.

a) 식물 조직으로부터 물질 추출, 발효, 정제, 분석까지의 프로세스
시료 준비

약 5g의 동결건조(lyophilized) 식물 조직에서 실험이 시작됩니다.

식물 조직은 여러 번의 물 추출(water extraction)을 거쳐 1차로 단순당(sugars) 등을 용출시킵니다.

발효(Fermentation)

추출액 또는 추출한 간단한 당류 등을 이용해 발효 단계를 진행합니다.

발효 과정에서 특정 미생물(효모, 세균 등)이 식물성 추출물 중 탄수화물·페놀류 성분을 변환하거나, 새로운 대사산물을 생산할 수 있습니다.

수지(resin) 정제

PVP, C18 수지 등을 사용해 발효액이나 추출액에서 바람직하지 않은 혹은 특정 군의 물질(예: 폴리페놀, 착색 성분 등)을 선택적으로 흡착/제거하거나, 원하는 물질만 분획(fraction)합니다.

이 단계는 2번 정도 반복되며, 정제된 분획물은 이후 기능성 분석이나 구조 분석에 사용됩니다.

분석 및 기능 평가

(1) 당류 정량 (예: 이온 크로마토그래피, HPAE-PAD)

(2) 탄수화물 전체 함량(Anthrone법 등)

(3) 페놀계 화합물 총 함량(Folin–Ciocalteu 방법)

(4) 특이 분자들의 LC–Q/TOF-MS 분석(정확질량분석) 등을 통해 어떤 기능성/활성 물질(예: oligosaccharides, phenolics)이 포함되어 있는지 확인합니다.

최종 기능성 평가

식물성 올리고당(HMOs 유사물질?)일 수도 있고, 또는 특정 분획된 탄수화물·폴리페놀 복합체가 기능성(예: 항산화, 프리바이오틱 효과 등)을 지닌지 시험할 수 있습니다.

요약하자면, 그림 (a)의 상자 안에 **“추출 → 발효 → 수지 정제 → 분석”**으로 이어지는 전체 워크플로우가 순서대로 나열되어 있으며, 각 단계에서 사용되는 정제 기법·분석 방법이 표시되어 있습니다

(b) 발효물 또는 정제 분획의 시간에 따른 반응 그래프
X축: 시간(시간 단위)

실험이 진행된 총 시간(예: 수십 시간 ~ 며칠).

미생물 생장 곡선 혹은 반응 진행 정도(예: OD, 흡광도, BLI(바이오레이어 간섭측정) 신호 등)를 수시로 측정하여 플롯.

Y축: 정규화된 데이터(‘Normalized OD’ or ‘Normalized response’)

보통 광학 밀도(OD), 굴절률 변화(BLI), 형광 세기 등 어떤 측정값을 0~1 범위 등으로 정규화시킨 값입니다.

그래프에 여러 색깔 곡선이 표시되어 있어, 서로 다른 조건(예: 다양한 균주, 다른 pH/온도, 또는 서로 다른 분획물 처리군)의 반응 차이를 비교하는 것으로 보입니다.

결과 해석

예를 들어, 파란색 곡선 “BLI 15h97 pHMO”나 노란색 곡선 “BAL 27536 pHMO” 등은 특정 시료/조건에서 미생물(또는 검출 센서)의 반응 속도 혹은 활성 변화를 나타낼 수 있습니다.

시간이 지남에 따라 곡선이 상승하면(↑), 미생물 증식률 증가, 혹은 센서 응답이 증가(예: 분자 상호작용 강화)가 일어났다는 의미일 수 있습니다. 반대로 꺾이거나 유지되면 성장이 정체 또는 상호작용이 일정함을 의미할 수 있습니다.

결론
그림 (a)는 식물조직으로부터 기능성 화합물을 추출·발효·정제·분석까지 이어지는 전체 연구 프로토콜을, 그리고 그림 (b)는 **그 최종 분획(또는 발효물)을 특정 조건에서 시간 경과에 따라 측정한 반응(미생물 성장, 분자 결합 반응 등)**을 그래프로 시각화한 것입니다. 이를 통해 연구자는 분획물의 조성(단당류, 올리고당, 페놀류)과 기능성(미생물 증식, 분자 간 상호작용 등)을 종합적으로 파악하여, 식물 발효 기반 기능성 물질의 효능 및 작용 기전을 평가할 수 있습니다.

a, Workflow of extraction, purification and characterization of HMOs from N. benthamiana leaves. b, Growth curves of HMO-consuming (BLI 15697) and control (BAL 27536) strains in media supplemented with HMO isolated from breast milk (HMO) or HMOs isolated from plants (pHMO). Error bars represent standard deviation.

a, N. benthamiana 잎에서 HMO의 추출, 정제 및 특성화의 작업 흐름.

b, 모유에서 분리된 HMO(HMO) 또는 식물에서 분리된 HMO(pHMO)를 보충한 배지에서 HMO를 섭취하는 균주(BLI 15697)와 대조군(BAL 27536)의 성장 곡선. 오차 막대는 표준 편차를 나타냅니다.

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Economic viability of plant-based HMO production

Plant-based HMO production can be commercially viable if it demonstrates cost-competitive or cost-advantage with current state-of-the-art production routes. To assess the economic viability of HMO production in a commercially relevant crop, we developed process models and compared the cost of HMO production in plants and microbes. To do this, we performed technoeconomic analysis (TEA) of the theoretical production of LNFPI. In the plant system, we adopted typical cellulosic biorefinery design using biomass from sorghum to coproduce HMOs along with biofuel because coproducing value-added bioproducts in biorefineries is a promising approach to maximize the use of biomass and hence improve the economics of biorefineries39,40. We assume that biomass sorghum can accumulate 0.31% dry weight of LNFPI in the entire biomass, as this was our highest yield following purification of LNFPI (Supplementary Table 4). We also developed process models and conducted TEA for the HMOs in Escherichia coli using the established processes and the highest reported yields of LNFPI41 from peer-reviewed papers at the time of conducting this TEA. The comparison between plant and microbial systems to produce the same product aims to provide indepth understanding of the cost–benefits of the systems.

TEA results indicate that producing LNFPI from the plant system is economically favourable compared to the microbial system (Supplementary Fig. 7). In the plant system, the minimum selling prices (MSPs) of LNFPI are US$4.9 kg−1 when selling ethanol at the cellulosic ethanol selling price and US$18.4 kg−1 when ethanol is sold at the target fuel price, respectively (Fig. 5). However, microbial-based LNFPI results in MSP of US$45.0 kg−1 with downstream recovery and purification being the largest cost contributor, followed by the cost of glucose (Supplementary Fig. 7b). The high cost obtained in the microbial system is largely due to its extremely low highest reported yields (0.48%) and recovery rate after bioconversion (62%). These results indicate that when microbial hosts are unable to produce HMOs at a comparable yield, plants may be a cost-effective bioplatform for high-value products. In addition to relative cost advantages of plant systems, using biomass as the feedstock to coproduce high-value compounds and biofuel offers considerable environmental benefits because biomass can absorb CO2 from the atmosphere during its growth42. Although current HMO yields in stable lines of the model plant N. benthamiana (Fig. 3) are below the yields in transiently expressing tissue, high yields could be achieved by optimizing the HMO constructs used for the production of stable lines, finding an optimal crop species for HMO production and optimizing growing conditions.

식물 기반 HMO 생산의 경제적 타당성

식물 기반 HMO 생산이 현재의 최첨단 생산 경로에 비해 비용 경쟁력이 있거나 비용 면에서 유리하다면 상업적으로 실행 가능할 수 있습니다. 상업적으로 관련된 작물에서 HMO 생산의 경제적 타당성을 평가하기 위해, 우리는 공정 모델을 개발하고 식물과 미생물에서 HMO 생산 비용을 비교했습니다. 이를 위해, 우리는 LNFPI의 이론적 생산에 대한 기술경제학적 분석(TEA)을 수행했습니다.

공장 시스템에서는

사탕수수를 원료로 하는 전형적인 셀룰로오스 바이오리파이너리 설계를 채택하여

바이오연료와 함께 HMO를 공동 생산했습니다.

바이오리파이너리에서 부가가치가 있는 바이오 제품을 공동 생산하는 것은 바이오매스의 사용을 극대화하고, 따라서 바이오리파이너리의 경제성을 향상시키는 유망한 접근 방식이기 때문입니다39,40. 우리는 바이오매스 사탕수수가 전체 바이오매스에서 0.31%의 건조 중량 LNFPI를 축적할 수 있다고 가정합니다. 이는 LNFPI 정제 후 가장 높은 수율이기 때문입니다(보충표 4). 또한, 우리는 확립된 공정과 당시 동료 심사 논문에 보고된 가장 높은 수율의 LNFPI41을 사용하여 대장균의 HMO에 대한 공정 모델을 개발하고 TEA를 수행했습니다. 동일한 제품을 생산하기 위한 식물과 미생물 시스템의 비교는 시스템의 비용 편익에 대한 심층적인 이해를 제공하는 것을 목표로 합니다.

TEA 결과는 식물 시스템에서 LNFPI를 생산하는 것이 미생물 시스템에 비해 경제적으로 유리하다는 것을 보여줍니다(보충 그림 7). 식물 시스템에서 LNFPI의 최소 판매 가격(MSP)은 셀룰로오스 에탄올 판매 가격으로 에탄올을 판매할 때 4.9달러/kg이고, 목표 연료 가격으로 에탄올을 판매할 때 18.4달러/kg입니다(그림 5). 그러나 미생물 기반 LNFPI의 경우, 다운스트림 회수 및 정제가 가장 큰 비용 기여 요인이고, 그 다음으로 포도당 비용이 뒤따르는 US$45.0kg−1의 MSP를 초래합니다(그림 7b). 미생물 시스템에서 발생하는 높은 비용은 주로 보고된 최고 수율(0.48%)이 매우 낮고, 생물학적 전환 후 회수율이 62%에 불과하기 때문입니다. 이 결과는 미생물 숙주가 비슷한 수율로 HMO를 생산할 수 없을 때 식물이 고부가가치 제품을 위한 비용 효율적인 바이오 플랫폼이 될 수 있음을 나타냅니다. 식물 시스템의 상대적 비용 이점 외에도, 바이오매스를 고부가가치 화합물과 바이오 연료를 공동 생산하는 원료로 사용하면, 바이오매스가 성장하는 동안 대기에서 CO2를 흡수할 수 있기 때문에 상당한 환경적 이점을 얻을 수 있습니다42. 현재 모델 식물 N. benthamiana(그림 3)의 안정된 계통에서 HMO 수확량이 일시적으로 발현되는 조직의 수확량보다 낮지만, 안정된 계통의 생산에 사용되는 HMO 구조를 최적화하고, HMO 생산에 적합한 작물 종을 찾고, 성장 조건을 최적화하면 높은 수확량을 달성할 수 있습니다.

Fig. 5: Plant-based platform improves the economics of producing the HMO, LNFPI.

Estimated MSP of LNFPI produced using biomass sorghum as a model production platform in two bioethanol price scenarios. Error bands represent final values calculated with ±20% of input parameters.

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Discussion

Human milk oligosaccharides are major contributors to the bioactive properties of human milk1,2. While the unique bioactivities of few HMOs have been investigated3,8,9,10,11, over 100 HMOs remain to be studied owing to lack of access to material, representing a wealth of potential bioactive molecules. Here, we report the production of all three classes of HMOs in planta. Notably, expression‎ of HMO biosynthetic pathways in planta created a variety of complex HMOs, including oligosaccharides that at present are not produced in microbial platforms, which indicates plants could serve as a platform for the production of a range of HMOs which are not currently producible in microbial hosts. Furthermore, we optimized the production of both specific HMOs and HMO classes by overexpressing nucleotide sugar biosynthetic pathways. The diversity of HMOs produced from inserting a relatively small number of genes shows the ability of plants to generate complex sugars. Transient expression‎ serves as a viable platform for testing HMO biosynthetic genes and small-scale production of HMOs for functional validation. Optimized l, laboratory-scale purification methods enabled the isolation of oligosaccharides, including HMOs, from plant tissue, showing the potential of plants as an industrial source of HMOs. HMOs purified from plants provided selective bifidogenic activity, indicating that they would serve as a potent prebiotic in vivo. Future pathway engineering could yield biosynthetic pathways for the production of all HMOs present in human milk, including complex, branched HMOs. This would enable the study and potential consumption of bioactive HMOs that are currently unavailable.

Despite the field of plant synthetic biology being in its nascent stages, plants have the potential to produce compounds of interest at lower costs than microbial platforms because of their intrinsic and unique metabolic capabilities39. Additionally, plants are capable of using atmospheric carbon dioxide during their growth cycle to produce target compounds and biofuels, improving the sustainability of target compound production. We demonstrate the ability of stably transformed plants to produce two HMOs that are abundant in breast milk of most mothers, 2′FL and LNFPI. While the feasibility of purifying HMOs from plants at industrial scales still needs to be validated, our TEA results indicate that HMO production in commercially relevant crops has the potential to be a more cost-effective platform than microbial production for complex HMOs.

As the demand for HMOs increases because of the growing infant formula and adult prebiotic markets, plants may emerge as a cost-competitive and sustainable platform for the production of diverse HMOs at agricultural scales. Furthermore, the diversity of plant-produced HMOs will provide researchers with access to HMOs that were previously inaccessible. Since the structure of an HMO determines its bioactivity, this could lead to the discovery of HMOs that treat various gastrointestinal illnesses. Additionally, production of HMOs in plants could permit direct consumption as food by directly ingesting the plant or products made from the plant. Such a product may serve as a consumable source of prebiotic HMOs for humans or be added to forage crops for animal consumption. Overall, the production of HMOs in planta provides the opportunity to simultaneously improve the scale of HMO production and expand the diversity of HMOs available to improve the gastrointestinal health of infants and adults.

토론

인간 모유 올리고당은 인간 모유의 생체 활성 특성에 주요 기여를 합니다1,2.

몇몇 HMO의 독특한 생체 활성이 연구된 바 있지만3,8,9,10,11,

100개가 넘는 HMO가 아직 연구되지 않았습니다.

여기에서는

식물에서 세 가지 종류의 HMO가

모두 생산되는 것을 보고합니다.

특히, 식물체 내 HMO 생합성 경로의 발현은 현재 미생물 플랫폼에서 생산되지 않는 올리고당을 포함한 다양한 복합 HMO를 생성했으며, 이는 식물이 현재 미생물 숙주에서 생산할 수 없는 다양한 HMO 생산을 위한 플랫폼 역할을 할 수 있음을 나타냅니다. 또한, 우리는 뉴클레오티드 당 생합성 경로를 과발현하여 특정 HMO와 HMO 클래스 모두의 생산을 최적화했습니다. 비교적 적은 수의 유전자를 삽입하여 생산된 HMO의 다양성은 식물이 복잡한 당을 생성할 수 있는 능력을 보여줍니다. 일시적 발현은 HMO 생합성 유전자를 테스트하고 기능 검증을 위한 HMO의 소규모 생산을 위한 실행 가능한 플랫폼 역할을 합니다. 최적화된 실험실 규모 정제 방법을 통해 식물 조직에서 HMO를 포함한 올리고당을 분리할 수 있었으며, 이는 식물이 HMO의 산업적 원천이 될 수 있는 가능성을 보여줍니다. 식물에서 추출한 HMO는 선택적 비피더스균 활동을 제공하여 생체 내에서 강력한 프리바이오틱 역할을 할 수 있음을 나타냅니다. 미래의 경로 공학은 복합 분지형 HMO를 포함하여 모유에 존재하는 모든 HMO의 생합성 경로를 생성할 수 있습니다. 이를 통해 현재 이용이 불가능한 생체 활성 HMO의 연구와 잠재적 소비가 가능해질 것입니다.

식물 합성 생물학 분야가 초기 단계에 있지만, 식물은 고유한 대사 기능 덕분에 미생물 플랫폼보다 저렴한 비용으로 관심 있는 화합물을 생산할 수 있는 잠재력을 가지고 있습니다39. 또한, 식물은 성장 주기 동안 대기 중의 이산화탄소를 사용하여 목표 화합물과 바이오 연료를 생산할 수 있어 목표 화합물 생산의 지속 가능성을 향상시킵니다. 우리는 대부분의 산모의 모유에 풍부하게 함유되어 있는 2가지 HMO(2′FL과 LNFPI)를 안정적으로 생산할 수 있는 식물의 능력을 입증했습니다. 식물을 통해 산업적 규모로 HMO를 정제하는 것이 가능한지는 아직 검증해야 하지만, 우리의 TEA 결과는 상업적으로 중요한 작물에서 HMO를 생산하는 것이 복잡한 HMO를 미생물로 생산하는 것보다 비용 효율적인 플랫폼이 될 수 있다는 가능성을 보여줍니다.

유아용 조제분유와 성인용 프리바이오틱스 시장의 성장으로 인해 HMO에 대한 수요가 증가함에 따라, 농경 규모에서 다양한 HMO를 생산할 수 있는 비용 경쟁력이 있고 지속 가능한 플랫폼으로 식물이 부상할 수 있습니다. 또한, 식물에서 생산되는 HMO의 다양성은 연구자들에게 이전에는 접근할 수 없었던 HMO에 대한 접근성을 제공할 것입니다. HMO의 구조가 생체 활성을 결정하기 때문에, 다양한 위장 질환을 치료하는 HMO가 발견될 수 있습니다. 또한, 식물에서 HMO를 생산하면 식물이나 식물에서 만든 제품을 직접 섭취함으로써 식품으로 직접 섭취할 수 있습니다. 이러한 제품은 인간을 위한 프리바이오틱 HMO의 소모성 공급원 역할을 하거나 동물 사료 작물에 첨가될 수 있습니다. 전반적으로, 식물에서 HMO를 생산하면 HMO 생산 규모를 확대하고 HMO의 다양성을 확대하여 유아와 성인의 위장 건강을 개선할 수 있습니다.

Methods

Plant growth

N. benthamiana was grown from seed in 3.5 inch square pots in a controlled environment facility. Plants were grown with a 12 h/12 h day/night cycle at ~700 µmol of photons per m2 s−1. Daytime growth chamber temperatures were kept at 26 °C. Night-time growth chamber temperatures were kept at 25 °C. Relative humidity in the growth chamber was kept between 60% and 75%.

Plasmid construction and transient expression‎

For transient expression‎, the native sequences for candidate glycosyltransferases were PCR amplified and cloned into the binary vector, PMS057 (ref. 43), using Golden Gate assembly44, Gibson assembly45 or restriction-ligation (see Supplementary Table 5 for sequences). XL1-blue E. coli cells were transformed with the assembled plasmids via heat shock46. Transformed cells were selected by plating cells on Lysogeny broth (LB) agar plates containing 50 µg ml−1 of kanamycin. Plasmid assembly was confirmed by means of miniprep and Sanger sequencing (Azenta). A. tumefaciens str. GV3101 was transformed using sequence-verified plasmids by electroporation47. Transformed colonies were selected using LB agar plates containing 50 µg ml−1 of kanamycin, 50 µg ml−1 of rifampicin and 10 µg ml−1 of gentamicin. A. tumefaciens str. GV3101 harbouring individual candidate glycosyltransferases were grown in LB overnight to OD600 nm (VWR, V-1200) of 0.8–1.2. The cultures were centrifuged at 4,000g for 10 min and the supernatant was decanted. Cell pellets were resuspended in infiltration media (10 mM MES, 10 mM MgCl2, 500 µM acetosyringone, pH 5.6) and incubated at room temperature for 1 h with gentle rocking (Thermolyne, VariMix). A. tumefaciens strains harbouring each glycosyltransferase were mixed in equal amounts alongside a strain harbouring the p19 silencing suppressor48 to reach a final OD600 nm of 0.5. A. tumefaciens mixtures were injected into the abaxial side of a leaf on a 4-week-old N. benthamiana using a needleless syringe. Each experiment was performed with three biological replicates.

For the production of stable lines, HMO10 and HMO11 constructs were generated through a multipart Golden Gate assembly containing subcloned transcriptional units. Assembled plasmids were transformed and sequence verified as described above. N. benthamiana was transformed using A. tumefaciens str. EHA105 harbouring HMO10 or HMO11 by the UC Davis Plant Transformation Facility.

Quantitative PCR with reverse transcription

Total messenger RNA was extracted using E.Z.N.A. plant RNA kit (Omega Bio-tek) following manufacturer’s directions using the RB lysis buffer variation and on-column DNase digestion; complementary DNA synthesis was achieved with SSIV Vilo IV kit using random hexamers (Thermo Fisher Scientific). Quantitative PCR was performed using a CFX96 Real-Time thermocycler (Bio-Rad) programmed for detection of SYBR intercalating dye with the following temperature programming: 95 °C for 3 min, then 95 °C for 30 s, 60 °C for 45 s, repeated 34 times, then a gradual increase from 65 °C to 95 °C at 0.5 °C per minute to generate melt curves. Sso-Advanced Universal SYBR Green Supermix (Bio-Rad) was used for qPCR amplification. A previously validated primer set was used to amplify EF1α for internal normalization, primers for target genes were designed with Benchling’s qPCR primer design wizard and synthesized by IDT. One target gene from each transcriptional unit was chosen for both constructs (Supplementary Table 6). Melt curves for the product of all primer sets were unimodal and steep, suggesting only a single product was formed for each primer set. No reverse-transcriptase controls showed no amplification within the dynamic range of samples, confirm‎ing the efficacy of DNAse treatment and no template controls instituted at the beginning of RNA extraction with no plant matter and kept in parallel with real samples throughout all molecular steps did not amplify, confirm‎ing lack of contamination with extraneous DNA. Normalized relative expression‎ was calculated using the ∆∆Cq method and normalized by setting the average level of amplification in the wild-type samples as 1.

HMO extraction for identification of HMOs from individual leaves

N. benthamiana leaves transiently expressing HMO biosynthetic enzymes were harvested 5 days after infiltration. Three leaves of N. benthamiana stable lines transformed with HMO10 and HMO11 were harvested at 4 weeks old. Following harvest, vasculature was removed and leaves were frozen in liquid nitrogen and lyophilized (Labconco, Freezone 4.5) for 2 days. Lyophilized leaves were homogenized via a bead mill (Retsch, MM400) at 20 Hz for 10 min. Oligosaccharides were extracted from 20 mg of lyophilized leaf tissue by ethanol precipitation. To each sample, 1 ml of 80% ethanol was added before homogenization on a bead mill at 10 Hz for 1 min. Samples were then precipitated overnight at −20 °C and centrifuged at 10,000g for 15 min. The supernatant was transferred to a 2 ml screw-cap tube. The pellet was washed twice by adding 500 μl of 80% ethanol, homogenizing via bead mill for 1 min and centrifuging at 10,000g for 15 min. The supernatant and washes were combined and dried in a vacuum centrifuge (Genevac EZ-2, SP Scientific). Dried supernatants were reconstituted in 200 μl of water and subjected to both C18 and PGC SPE (Thermo Fisher Scientific) in 96-well plate format. C18 cartridges containing 25 mg of stationary phase were first conditioned by two additions of 250 μl of acetonitrile (ACN) followed by four additions of 250 μl of water. Samples were then loaded and eluted with two volumes of 200 μl of water. PGC cartridges containing 40 mg of stationary phase were conditioned by addition of 400 μl of water, 400 μl of 80% (v/v) ACN and water, followed by two volumes of 400 μl of water. The sample eluate from C18 SPE was then loaded, washed thrice with 500 μl of water and eluted using two volumes of 200 μl of 40% (v/v) ACN and water. The purified extracts were dried in a vacuum centrifuge and reconstituted in 100 μl of water before injecting 5 μl for liquid chromatography mass spectrometry (LC–MS) analysis.

For quantification of LNFPI in Figs. 2 and 3, LNFPI at known concentrations was added to the extraction solution. The extraction solution was then used on wild-type N. benthamiana. This was done to ensure accuracy of HMO quantification by accounting for HMO losses in the extraction processes and ion suppression that could occur due to the plant metabolites present.

LC–MS analysis of HMOs from individual leaves

For initial screening, chromatographic separation was carried out using a Thermo Scientific Vanquish UHPLC system equipped with a Waters BEH C18 Amide column (HILIC) (1.7 µm, 100 mm × 2.1 mm). A 10 min binary gradient was used based on ref. 49: 0.0–4.0 min, 25–35% A; 4.0–8.50 min, 35–65% A; 8.50–8.70 min, 25% A. Mobile phase A consisted of 3% ACN (v/v) in water with 0.1% formic acid and mobile phase B consisted of 95% ACN (v/v) in water with 0.1% formic acid.

For identification of HMOs produced, we performed LC–MS analysis using a Thermo Scientific Vanquish 3000 UPLC system connected to Thermo Scientific Q Exactive mass spectrometer. Chromatographic separation was carried out using a Hypercarb PGC column (5 µm, 150 mm × 1 mm, Thermo Scientific). A 40 min binary gradient using 3% ACN in water containing 0.1% formic acid (Solvent A) and 90% (v/v) ACN in water containing 0.1% formic acid was performed as follows: 100% A, 0–2.5 min; 100–84% A, 2.5–15 min; 84–42% A, 15–20 min; 42–0% A, 20–22 min; 0% A, 22–28 min; 0–100% A, 28–30 min; 100% A, 30–40 min.

For identification of HMOs, the Q Exactive mass spectrometer equipped with an electrospray ionization source was operated in positive ionization mode with the following parameters: scan range m/z 133.4–2,000; spray voltage 2.5 kV, capillary temperature 320 °C, aux gas heater temperature 325 °C, sheath gas flow rate 25, aux gas flow rate 8, sweep gas flow rate 3. MS/MS analysis was performed using stepped collision energies of 20, 30, 40 eV. MsDIAL was used for data analysis50.

For quantification of LNFPI and HMO profiling, mass spectral analysis was carried out on an Agilent 6530 Accurate-Mass Q-ToF MS operated in positive mode using data-dependent acquisition. The gas temperatures were held at 150 °C. The fragmentor, skimmer, octopole and capillary were operated at 70, 55, 750 and 1,800 V, respectively. The collision energy was based on the empirically derived linear formula (1.8 × (m/z/100) − 3.6). The reference mass used for calibration was m/z 922.009798. The Agilent MassHunter Qualitative software was used for data analysis. Oligosaccharides were identified using an inhouse library, their MS/MS spectra and comparison to either authenticated standards or a pool of HMOs of known composition.

Extraction and purification of HMOs from pooled leaves

Five grams of lyophilized and ground N. benthamiana leaves transiently expressing the LNFPI and GDP-fucose biosynthetic pathways was mixed with 150 ml of water and agitated for 15 min at room temperature in a stirring plate. The mixture was centrifuged at 4,000g for 5 min and the supernatant was separated. The extraction was repeated two more times, combining the supernatant each time. The final supernatant was filtered using a 0.22 µm Millipore Steritop vacuum filter. The extraction process was carried out in duplicate to ensure reproducibility.

Yeast fermentation was carried out to eliminate simple sugars (glucose, sucrose and fructose) from the extracts51. Briefly, autoclaved extracts were inoculated with 0.4 g l−1 of commercial active dry yeast Saccharomyces cerevisiae52 (UCD 522 Montrachet, Lallemand) at 30 °C, 150 rpm for 24 h (Excella E24 Incubator Shaker Series, New Brunswick Scientific). After 24 h, the samples were centrifuged at 4,000g for 5 min and filtered using a 0.22 µm Millipore Steritop vacuum filter to remove the yeast. Samples were concentrated using a vacuum concentrator (Genevac miVac Centrifugal Concentrator) at room temperature and frozen until their purification.

PVPP (Sigma-Aldrich) was used to bind phenolic compounds within the sample following a previous protocol52. Briefly, 3 g of PVPP was conditioned by mixing it with 100 ml of 12 M HCl at 100 °C for 30 min with constant stirring in a stirring plate. After cooling off, the slurry was centrifuged at 4,000g for 5 min and filtered using a 0.22 µm Millipore Steritop vacuum filter. Subsequently, the PVPP was washed with nanopure water until the flow-through reached pH 7. Activated PVPP was mixed with water to a final concentration of 20 mg of PVPP per ml.

PVPP suspension was added to the extracts at a concentration of 6 mg of PVPP per ml and agitated at room temperature for 15 min on a stirring plate. After the time had elapsed, the sample containing the plant extracts and the PVPP was centrifuged at 4,000g for 5 min and filtered using a 0.22 µm Millipore Steritop vacuum filter to separate the PVPP containing the bound phenolics. To eliminate residual phenolics from the extracts, more PVPP was added to the supernatant (6 mg of PVPP per ml) and the process was repeated. The filtrate containing the oligosaccharides was concentrated and frozen until further purification.

Two SPE columns of 60 ml were packed with 15 g of Bondesil-C18, 40 µm suspended in 20 ml of ACN. After the ACN was drained, a frit was added to the C18. Before loading the samples, the columns were conditioned with three volumes of ACN and three volumes of nanopore water. PVPP-treated extracts were loaded onto the conditioned C18 columns and the oligosaccharides were washed with 250 ml of nanopure water divided into four washes. To ensure the complete removal of interfering compounds, C18 SPE was repeated two more times. The purified HMO fractions were dried in a vacuum concentrator (Genevac miVac Centrifugal Concentrator) at room temperature and frozen.

Compositional analysis of plant material

Total carbohydrate content was assessed by the anthrone method with modifications53. In a 96-well microplate, 40 μl of purified and diluted extracts were combined with 100 μl of anthrone reagent (2 mg ml−1 (w/v) in cold 98% sulfuric acid) and mixed through pipette tip aspiration. The microplate was incubated for 3 min at 92 °C in a water bath followed by 5 min at a room temperature water bath and then 15 min in a 45 °C Thermolyne Benchtop muffle furnace (Thermo Fisher Scientific). The plate was cooled for 3 min at room temperature before measuring the absorbance with a SpectroMax M5 UV/Vis spectrophotometer (Molecular Devices) at 630 nm. Total carbohydrate quantification calculations were based on a glucose standard curve. Each plant extract was prepared in duplicate and each sample was further analysed in duplicate.

Total phenolic content of the extracts was determined according to the Folin–Ciocalteu spectrophotometric method as described by ref. 54.

Simple sugars (glucose, sucrose and fructose) were quantified by high-performance anion exchange chromatography with pulsed amperometric detection on a Thermo Fisher Dionex ICS-5000+ HPAE-PAD system based on a method described by ref. 55 with modifications. Diluted extracts (1:100, v/v or 1:1,000 in nanopure water) were filtered through a 0.2 mm syringe filter (Agilent Captiva Econo Filter, PES, 13 mm, 0.2 μm) into 2 ml vials with septa. The samples (25 μl) were injected into a CarboPac PA200 guard column (3 × 50 mm) and a CarboPac PA200 analytical column (3 × 250 mm) and chromatographic separation was carried out with a 12 min gradient elution (from 0.6% to 25% B in 12 min), 0.5 ml min−1 flow rate. The solvent system consisted of A: 100% water; and B: 200 mM sodium hydroxide. Calibration curves (correlation coefficient ≥0.999) were prepared using glucose, sucrose and fructose standards.

Quantification of HMOs by QqQ LC–MS

Detection and quantitation of HMOs were performed using an Agilent 6470 triple quadrupole LC–MS system (QqQ LC–MS) equipped with an Advance Bio Glycan Map column (2.1 mm × 150 mm, 2.7 μm, Agilent). The mobile phase consisted of 10 mM ammonium acetate in 3% ACN, 97% water (v/v, pH 4.5; A) and 10 mM ammonium acetate in 95% ACN, 5% water (v/v, pH 4.5; B). The chromatographic separation was carried out at 35 °C with gradient elution at a flow rate of 0.3 ml min−1. The MS analysis was conducted in positive ion mode with source parameters as follows: the gas temperature was 150 °C at a flow rate of 10 l min−1; the nebulizer was 45 psi; the sheath gas temperature was 250 °C at a flow rate of 7 l min−1; capillary voltage was 2,200 V. See Supplementary Table 2 for gradient and multiple reaction monitoring transitions.

Characterization of HMOs by LC-QToF-MS

Oligosaccharides were purified by a two-step SPE using C18 (HyperSep C18–96, 50 mg bed weight; Thermo Fisher Scientific) and PGC (HyperSep Hypercarb-96, 25 mg bed weight; Thermo Fisher Scientific)56. The samples were filtered (Captiva Premium Syringe Filter PES membrane, 4 mm diameter, 0.2 µm pore size, LC/MS certified) into 200 µl vials.

Individual oligosaccharide compositions were analysed with an Agilent 6520 NanoChip LC-QToF mass spectrometer. Oligosaccharides separation was achieved with a microfluidic high-performance liquid chromatography PGC chip containing an enrichment (4 mm, 40 nl) and an analytical (75 μl × 43 mm) column as well as a nanoelectrospray tip, using a binary solvent gradient of solvent A (5 mM ammonium acetate in 3% ACN, 97% water (v/v)) and solvent B (5 mM ammonium acetate in 90% ACN, 10% water (v/v)) based on a previously optimized method55. The gradient was 0–16% B at 0–20 min, 16–44% B at 20–30 min, 44–100% B at 30–35 min, 100% B at 35–45 min and 100–0% B from 45 to 45.1 min, followed by a 15 min re-equilibration of 100% A57. The mass spectrometer was operated in positive ionization mode with a range of m/z 320–2,500 and an electrospray capillary voltage of 1,800–1,900 V. Reference masses of m/z 922.009 and 1,221.991 provided continuous internal calibration. All samples were analysed using MS/MS with tandem fragmented peaks selected by the automated precursor selection of the six ions with highest signal intensity with a medium isolation width. The Q-ToF MS had a ramped collision energy slope of 0.02 based on m/z values with an offset of −3.5 V. The acquisition rate of 1 spectrum per s was used for both MS and MS/MS. Each spectrum was manually examined and molecular masses were confirmed with Agilent MassHunter Qualitative Analysis B.07.00 software using the molecular feature extraction and a maximum tolerance of 20 ppm.

Bacterial strains and growth conditions

B. longum subsp. infantis ATCC 15697 and B. animalis subsp. lactis ATCC 27536 were cultured at 37 °C in a Coy vinyl anaerobic bubble with an atmosphere of 2.5% H2, ~5% CO2 and balance N2. Routine culturing was done with Difco MRS + 0.05% l-cysteine HCl (MRSC) and carbohydrate-specific culturing was done with modified MRS (mMRSC), which was prepared per litre as follows: 10 g of Bacto proteose peptone no. 3, 10 g of Bacto casitone, 5 g of Bacto yeast extract, 2 g of triammonium citrate, 5 g of sodium acetate trihydrate, 200 mg of magnesium sulfate hexahydrate, 34 mg of manganese sulfate monohydrate, 0.5 g of l-cysteine HCl and 1.063 g of Tween-80. Normally, 2 g of anhydrous dipotassium phosphate would also be added but it was precipitated by the plant HMO preparation.

Growth curves

One colony was used to inoculate 1 ml mMRSC + dipotassium phosphate + 1% lactose monohydrate and incubated for 24 h. The growth curve inoculum was cultured by diluting the 24 h culture 1:100 in mMRSC + dipotassium phosphate + 1% lactose, then incubating for 12–15 h. The inoculum was prepared by washing the cells twice with one volume of 1× PBS. Growth curve cultures (160 μl) were contained in flat-bottomed, optically clear, 96-well, lidded plates and they had a final inoculum and sugar concentration of 1% in mMRSC. Lactose was the growth control substrate, water was the no-growth control substrate and pooled HMO58 was the HMO-growth control substrate. Cultures were done in triplicate. Wells were overlaid with 40 μl of sterile mineral oil and incubated in a BMG SpectroStar Nano. The plate reader was set to read the OD600 nm of each well 30 times in a spiral pattern every half-hour with medium orbital shaking before each read. All media had uninoculated controls whose OD600 nm was subtracted from that of the inoculated medium.

Technoeconomic analysis

In this study, we used SuperPro Designer v.12 to develop technoeconomic models of HMOs which can be produced from both plant and microbial systems. To do this, we first developed process models and then applied discount cash flow analysis of the theoretical production of LNFPI in plants and microbes. The simplified process flow diagram can be found in Supplementary Fig. 8. In the plant system, we adopted integrated cellulosic biorefinery design to coproduce HMO and ethanol to maximize the use of plant biomass. Biomass sorghum was used as the representative plant because its characteristics, such as high yields and drought tolerance, are ideal for biofuel production. Previous studies demonstrated that using biomass sorghum as the representative plant to accumulate value-added bioproducts could improve the economic performance of an integrated biorefinery39,40. Since ethanol is coproduced in the biorefinery, two ethanol selling prices were considered: (1) baseline cellulosic biofuel selling price of US$1.44 l−1 of gasoline equivalent and (2) target fuel price of US$1.00 l−1 of gasoline equivalent.

Briefly, biomass sorghum with 0.31% dry weight HMOs accumulated in the plant biomass are harvested and transported to the biorefinery gate for preprocessing and short-term onsite storage. HMO extraction, separation and recovery is then conducted on the basis of our laboratory processes and as described in previous texts. Water is used to extract HMO from the biomass (room temperature for 6 h) since the industrial extraction process will last longer than the laboratory-scale process given the large quantity of biomass being processed. The extraction efficiency is assumed at 90% based on previous studies39. After extraction, the slurry is first cooled down to room temperature and is sent to centrifugation to remove water. This water is sent to the wastewater treatment unit located in the biorefinery for recycling and reusing. Afterwards, multistage ultrafiltration is used to recover HMO from the extraction stream. Before transporting to HMO onsite storage, another centrifuge is applied to ensure the recovery and purity of the final product. In this plant system, the extracted HMOs are considered as the main product with the purity of >95%. The remaining biomass from biomass sorghum is routed to ionic liquid pretreatment for biomass deconstruction. After ionic liquid pretreatment, enzymatic hydrolysis and ethanol fermentation are conducted to produce ethanol, followed by distillation and molecular sieve to remove excess water. Wastewater from the overall process is routed to the wastewater treatment sector to produce reusable process water and biogas, which can be combusted in the onsite turbogenerator, along with other solids from biomass, to generate heat and electricity that can satisfy the facility’s need. In the microbial system, HMOs were produced as the single product in the biorefinery. Unlike the plant system, pure sugar is used in the microbial system as the sole feedstock and no wastewater treatment sector and onsite combustion are designed for the microbial system. The downstream processing of microbial production is adopted from peer-reviewed publication41. After LNFPI is produced in the bioreactor under the bioconversion condition of 30 °C for 52 h, ultrafiltration is first applied to remove biomass and ion exchange chromatography to remove ions and other charged impurities. Multistage nanofiltration is further used to reduce the total volume and remove excess impurities. Gel filtration is used for the final purification of HMO from the microbial production process. The purity of the final product is >98% through this process.

After developing technoeconomic models in SuperPro Designer, we performed mass and energy balance and then applied discounted cash flow analysis to quantify the MSP of HMOs (US$ kg−1). For both systems, we assume the biorefinery can operate 24 h per day and 330 days per year for 30 years. The unit price of biomass sorghum is assumed at US$95 per bone-dry tonne and we assume in the plant system the cellulosic biorefinery can intake 2,000 bone-dry tonnes of biomass sorghum per day. The unit price of ionic liquid is US$2 kg−1 with a range of US$1–5 kg−1. Total capital investment includes installed equipment cost, piping costs, engineering costs, warehouse, site development, construction fees, contingency costs, land costs, startup and working capital. Annual operating costs include raw materials costs, utility costs, labour costs and facility-dependent costs such as insurance. These parameters are kept constant as per the 2011 National Renewable Energy Laboratory report59.

Statistics and reproducibility

No statistical method was used to predetermine sample size as performing experiments in biological triplicate is standard of the field. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment. All experiments looking at individual leaves were conducted in biological triplicate. For the laboratory-scale purifications, extractions were completed in duplicate and each duplicate was measured with two technical replicates. Microbial growth assays were conducted in biological triplicate. For statistical analysis of LNFPI optimization, a heteroscedastic two-tailed Student’s t-test with the LNFPI pathway expressed alone was used as the reference group in RStudio v.1.2.5033. GraphPad v.10 was used to plot the microbial growth assays.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The main data supporting the findings of this study are available within the article and its Supplementary Information.

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