Engineering Microbial Cell Factories for the Production of Plant Natural
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Liu et al. Microb Cell Fact (2017) 16:125 DOI 10.1186/s12934-017-0732-7 Microbial Cell Factories REVIEW Open Access Engineering microbial cell factories for the production of plant natural products: from design principles to industrial‑scale production Xiaonan Liu1,2†, Wentao Ding1† and Huifeng Jiang1* Abstract Plant natural products (PNPs) are widely used as pharmaceuticals, nutraceuticals, seasonings, pigments, etc., with a huge commercial value on the global market. However, most of these PNPs are still being extracted from plants. A resource-conserving and environment-friendly synthesis route for PNPs that utilizes microbial cell factories has attracted increasing attention since the 1940s. However, at the present only a handful of PNPs are being produced by microbial cell factories at an industrial scale, and there are still many challenges in their large-scale application. One of the challenges is that most biosynthetic pathways of PNPs are still unknown, which largely limits the number of candidate PNPs for heterologous microbial production. Another challenge is that the metabolic fuxes toward the target products in microbial hosts are often hindered by poor precursor supply, low catalytic activity of enzymes and obstructed product transport. Consequently, despite intensive studies on the metabolic engineering of microbial hosts, the fermentation costs of most heterologously produced PNPs are still too high for industrial-scale produc- tion. In this paper, we review several aspects of PNP production in microbial cell factories, including important design principles and recent progress in pathway mining and metabolic engineering. In addition, implemented cases of industrial-scale production of PNPs in microbial cell factories are also highlighted. Keywords: Plant natural products, Synthetic biology, Microbial cell factories, Metabolic engineering, Industrial production Background threaten the supply of these natural products [4]. Te Tousands of plant natural products (PNPs) can be uti- yield of natural products from plants also cannot satisfy lized as drugs, cosmetics, dyes, seasonings, nutraceuti- the demands of the growing market. Due to the struc- cals, and industrial chemicals, all of which play important tural complexity of most natural products, total chemical roles in human life [1]. Unfortunately, in recent years synthesis approaches are often inefcient and accompa- over-exploitation has endangered more than 15,000 nied by large amounts of waste and heavy pollution [5]. medicinal plant species in their natural habitats, which Terefore, engineering microbial cell factories to produce resulted in a precarious and unsustainable supply of the these high-value PNPs has become a promising solu- invaluable natural products [2, 3]. At the same time, sea- tion to protect endangered plants and prevent pollution sonal, climatic or other environmental variations can also from chemical synthesis [4, 6]. Current developments in biological “omics” techniques and synthetic biology pro- vide excellent tools to remove obstacles in the construc- *Correspondence: [email protected] tion of microbial cell factories to produce PNPs [5, 7, 8]. † Xiaonan Liu and Wentao Ding contributed equally to this work Over the past few decades, there has been great progress 1 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China in multiple aspects of heterologous synthesis of PNPs, Full list of author information is available at the end of the article including the identifcation of biosynthetic pathways, the © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Microb Cell Fact (2017) 16:125 Page 2 of 9 construction of microbial cell factories, and the develop- 115 diverse cucumber lines [20, 21]. Whole-genome ment of industrial production [9]. Engineered microbial sequencing of Siraitia grosvenorii, combined with tran- cell factories have been successfully applied to produce scriptomic (RNA-Seq) and bioinformatic analyses made PNPs from renewable carbon sources at an industrial a great contribution to illuminate the biosynthetic path- scale. Further progress in the feld of microbial cell fac- way of mogroside V [22]. Glycosyltransferases of ginse- tories will not only potentially save endangered plants, nosides were also successfully cloned based on the ESTs but also profoundly change the traditional ways of PNP (Expressed Sequence Tags) and cDNA database of Panax production. ginseng [23, 24]. Six enzymes acquired by RNA-Seq from In this review, we summarize the recent progress of mayapple were assembled into the complete biosyn- microbial cell factory engineering, identifcation of novel thetic pathway of the etoposide aglycone [25]. Terefore, biosynthetic pathways, and the production of PNPs on “omics” technologies have greatly accelerated the pace of an industrial scale. Firstly, two tentative strategies based identifcation of novel genes from the biosynthetic path- on biological “omics” and synthetic biology technologies ways of PNPs. for gene mining from the biosynthetic pathways of PNPs are introduced, followed by a discussion of three piv- Recombined artifcial biosynthetic pathways otal steps in the optimization of microbial cell factories, Currently, various online databases providing enormous including the improvement of precursor supply, enzyme amounts of genomic data also facilitate the identifcation activity and product transport. Finally, several milestone of novel genes. For example, the 1000 plants project (1KP) examples of industrial PNP production in microbial cell proposed to collect the transcriptomes of 1000 plant spe- factories are showcased (Fig. 1). cies [26]. Te Medicinal Plant Genomics Resource pro- vides transcriptome and metabolome resources from Mining of PNP biosynthetic pathways medicinal plants [27]. Phytozome is a comparative plat- Te frst step in the construction of a PNP-producing form for green plant genomics, providing a view of the microbial cell factory is to identify the original biosyn- evolutionary history of each plant’s genes, as well as thetic pathway. Te rapid development of molecular access to the sequences and functional annotations [28]. biology, “omics” technologies and bioinformatics, has Moreover, based on detailed information about genes, enabled great breakthroughs in the identifcation of PNP- enzymes, reactions and their regulation, comprehensive biosynthetic pathways. In this section, recent approaches metabolic networks of PNPs have been composed based in gene exploration for the production of natural com- on reactions mined from diferent species using bioin- pounds are reviewed. formatics databases such as KEGG (Kyoto Encyclopedia of Genes and Genomes) [29]. From these databases, it is Sequencing‑guided pathway exploration therefore possible to “dig up” genes from diferent species Traditionally, reverse transcription-polymerase chain to construct artifcial biosynthetic pathways of PNPs for reaction (RT-PCR), rapid amplifcation of cDNA ends which the natural pathway is unknown. For instance, the (RACE) [10–12], RNA interference (RNAi) [13, 14], biosynthesis of opioids in yeast is considered to be one virus-induced gene silencing (VIGS) [15] and isotopic of the most remarkable landmarks of synthetic biology, tracer methods [16] had been used to uncover novel representing a highly sophisticated feat of engineering a genes in the biosynthetic pathways of PNPs. However, very complex metabolic pathway in microbe [30–32]. So these technologies are usually time-consuming, labori- far, 21 enzymes were successfully reconstructed in yeast ous, and consequently expensive. With the rapid devel- for the heterologous biosynthesis of opioids by combin- opment of sequencing technology, “omics” analysis and ing the building blocks derived from multiple species, high-throughput screening technologies, thousands of such as Papaver somniferum, Papaver bracteatum, Cop- functional genes related to PNP-biosynthetic pathways tis japonica, Eschscholzia californica, Rattus norvegicus, have been identifed in recent years [17]. For example, Pseudomonas putida, and yeast [31]. Additionally, rasp- the assembly of the Salvia miltiorrhiza transcriptome berry ketone [33], salidroside [34], gastrodin [35], and provided a valuable resource for the investigation of salvianic acid A (SAA) [36] were also successfully syn- the complete biosynthetic pathway of tanshinone [18]. thesized in microorganisms using recombined artifcial High-density genetic linkage mapping using recombi- pathways. Terefore, it has become feasible to construct nant inbred lines (RILs) had been used to decipher the the biosynthetic pathways of PNPs without genetic infor- genetic networks underlying favonoid biosynthesis mation from the original plant. Unprecedented microbial in Brassica napus [19]. Genes related to cucurbitacins synthesis of PNPs via recombined artifcial pathways pro- biosynthesis were characterized