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The aroma characteristics of Pu-erh tea were studied in this work. A total of 117 volatile compounds were identified by HS-SPME combined with GC–TOF/MS, of which 29 active aroma compounds were identified by olfactometry. The active aroma compounds of Pu-erh tea were analyzed by intensity aroma (IA), aroma extraction dilution (AEDA), sensory quantification value (MF), detection frequency (DFA) and odor activity value (OAV). 24, 21 and 23 active aroma components were identified by DFA, AEDA and OAV, while 20 by IA and MF. Recombination of the obtained active aroma components indicated that OAV method was best for determining the active aroma compounds as the aroma profile of OAV recombination model was the most similar to that of Pu-erh tea sample. Omission test of the OAV recombination model furtherly identified 19 characteristic active aroma compounds of Pu-erh tea, and linalool, 1,2,3-trimenthoxybenzebe, 1,2,4-trimethoxybenzene and ɑ-ionone were the key characteristic active aromatic compounds.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Summary The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non‐economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof‐of‐principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α‐bisabolene, β‐bisabolene and γ‐bisabolene through heterologous expression of the α‐bisabolene synthase from Abies grandis, the β‐bisabolene synthase gene from Zingiber officinale and the γ‐bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher‐level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products., To our knowledge, we have unprecedentedly verified the function of α‐bisabolene synthase, β‐bisabolene synthase and γ‐bisabolene synthase in Y. lipolytica and successfully employed the enzyme to synthesize bisabolene in Y. lipolytica for the first time. Our study reports for the first time the utilization of terpenoid efflux pumps to improve the secretion, and thus the production, of bisabolene in microbial cell factories. We demonstrated for the first time the conversion of waste cooking oil to bisabolene, which has important potential applications in waste management as well as economical and sustainable production of valuable bisabolene from waste feedstocks.

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y. lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Background In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Monoterpenoids are an important class of natural products that are derived from the condensation of two five‑carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.

BackgroundLimonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly.ResultsIn this proof-of-principle study, the oleaginous yeastYarrowia lipolyticawas successfully engineered to produce bothd-limonene andl-limonene by introducing the heterologousd-limonene synthase fromCitrus limonandl-limonene synthase fromMentha spicata, respectively. However, only 0.124 mg/Ld-limonene and 0.126 mg/Ll-limonene were produced. To improve the limonene production by the engineered yeastY. lipolyticastrain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis inY. lipolytica. Through the overexpression ofHMGRgene, the titers ofd-limonene andl-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineeredY. lipolyticastrains from glucose, and the final titers ofd-limonene andl-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved ford-limonene andl-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineeredY. lipolyticastrains was investigated. We showed thatd-limonene andl-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization.ConclusionsThis study represents the first report on the development of a new and efficient process to convert waste cooking oil intod-limonene andl-limonene by exploiting metabolically engineeredY. lipolyticastrains for fermentation. The results obtained in this study lay the foundation for more future applications ofY. lipolyticain converting waste cooking oil into various industrially valuable products.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast

In recent years, adaptive laboratory evolution (ALE) has emerged as a powerful tool for basic research in microbiology (e.g., molecular mechanisms of microbial evolution) and efforts on evolutionary engineering of microbial strains (e.g., accelerated evolution of industrial strains by bringing beneficial mutations). The ongoing rapid development of next-generation sequencing platforms has provided novel insights into growth kinetics and metabolism of microbes, and thus led to great advances of this technique. In this review, we summarize recent advances in the applications of long-term and short-term ALE techniques mainly for microbial strain engineering, and different modes of ALE are also introduced. Furthermore, we discuss the current limitations of ALE and potential solutions. We believe that the information reviewed here will make a significant contribution to further advancement of ALE.适应性实验室进化 (Adaptive laboratory evolution，ALE) 技术已成为微生物学基础研究和工业微生物育种的强大工具，被广泛用来研究影响菌株表型、性能和稳定性的进化潜力以及快速获取含有有益突变的工业生产菌株。近年来，随着基因组测序技术的进步，关于微生物新陈代谢机理和动力学方面的研究变得更加广泛和深入，这也极大促进了适应性实验室进化技术的快速发展。文中主要介绍了长期、短期适应性实验室进化技术在微生物育种方面的应用实例，并总结归纳了该技术在快速高效构建优良菌株过程中的方式与作用。最后分析了目前ALE技术面临的瓶颈问题及其可能的解决方法，以期能够为该技术的未来发展提供有价值的参考依据。.

Background: The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders their isolation from plant sources economically inviable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene.Results: In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, β-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the β-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus, respectively. Subsequently, metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize de novo bisabolene production by the engineered Y. lipolytica strains from glucose. Our engineering strategies have led to engineered Y. lipolytica strains that produce 282.6 mg/L α-bisabolene, 48.3 mg/L β-bisabolene and 5.3 mg/L γ-bisabolene. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from waste cooking oil. The results showed that α-bisabolene, β-bisabolene and γ-bisabolene could be produced at the respective titers of 973.0 mg/L, 68.2 mg/L, 20.2 mg/L in shake flasks. These titers correspond to 2433-fold, 340-fold and 404-fold enhancement in bisabolene production, respectively, over the parent strain.Conclusions: To our knowledge, this is the first report of bisabolene production in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (t LS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W−N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (t NDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W−N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a t LS–t NDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Carboxylic acids contain carboxyl groups that can undergo a wide range of chemical transformation. Therefore, they serve as key platform chemicals for the production of high value-added industrial products. Currently, the majority of carboxylic acid platform chemicals is produced predominantly through traditional chemical synthesis. However, these chemical conversion processes are heavily dependent on fossil fuels and often lead to serious environmental pollution. Recently, the rapid development in metabolic engineering of microbes provide a new and promising alternative route for producing carboxylic acids as platform chemicals. We envision that these bio-based manufacturing processes using microbial cell factories will help move the industrial production of carboxylic acid platform chemicals towards a more sustainable, environmentally friendly and economically competitive direction. While Escherichia coli and Saccharomyces cerevisiae have been the workhorses for biochemical production through metabolic engineering, non-conventional microbes are emerging as suitable hosts for producing carboxylic acids to meet the needs of the industries. Here, we review the employment of metabolic engineering strategies on non-conventional microbes to serve as microbial cell factories for the production of industrially important carboxylic acid platform chemicals.

Additional file 1: Table S1. Information on fatty acid composition of the commercial vegetable oil and waste cooking oil. Table S2. Primers used in PCR. Figure S1. Inhibitory effects of d‑limonene and l-limonene on S. cerevisiae AY15 and Y. lipolytica Po1g KU70Δ. Figure S2. GC–MS analysis of solvent overlay-extracted limonene from engineered strains cultures. Figure S3. Effect of different carbon sources on growth of Y. lipolytica. Figure S4. Effect of addition of glucose on biosynthesis of limonene. Figure S5. The OD600 values of different engineered Y. lipolytica strains cultured in YPD medium. Figure S6. Effect of temperature on volatilization of limonene. Figure S7. The pHs during fermentation of the engineered Y. lipolytica Po1g KdHR and Po1g KlHR strains in a 250-mL shake flask. Figure S8. The data of limonene batch-fermentation parameters in the 5 L-fermenter. Figure S9. Effect of Mg2+ on growth of Y. lipolytica. Figure S10. Effect of Mn2+ on d-limonene or l-limonene accumulation in Po1g KdHR or Po1g KlHR. Figure S11. Effect of concentration of peptone on the production of d-limonene and l-limonene. Figure S12. The data of limonene fed-batch fermentation parameters in the 5-L fermenter. Figure S13. Map of the plasmid pYLEX1. Figure S14. Map of the recombinant plasmid pYLdLS. Figure S15. Maps of the overexpression recombinant plasmid pYLA1 and pYLdA1. Figure S16. The GC–MS analysis of fatty acid composition in waste cooking oil and commercial vegetable oil.

Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

Background Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. Results The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 μg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 μg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 μg/L and 110 μg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. Conclusions This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica. Electronic supplementary material The online version of this article (10.1186/s12934-018-1014-8) contains supplementary material, which is available to authorized users.

Limonene (C₁₀H₁₆) and bisabolene (C₁₅H₂₄) are both naturally occurring terpenes in plants. Depending on the number of C₅ units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.柠檬烯和红没药烯均为植物天然产物，分别属于单萜类和倍半萜类化合物，能够预防和治疗癌症等多种疾病。以其作为前体物，还可以转化合成多种具有高附加值的工业产品，例如药品、保健品、化妆品及生物燃料等。目前柠檬烯和红没药烯的工业生产主要是通过植物提取法实现的，但从植物组织中提取柠檬烯和红没药烯存在着产物含量低和分离纯化困难等缺点。微生物代谢工程的快速发展为这些植物天然产物的生产提供了一条更具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些有价值的植物天然产物的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。文中系统综述了近年来代谢工程技术在微生物合成柠檬烯和红没药烯过程中的应用进展，包括所涉及的宿主菌株、关键酶、代谢途径及其改造等，并探讨了其未来发展方向。.

Additional file 1: Table S1. Primers used for the construction of the heterologous CoA-dependent pathway. Figure S1. GC/MS detection of 1-butanol production in two Y. lipolytica engineered strains carrying the chromosomally integrated plasmid pYLEX1-All-GPD or pYLEX1-All-MDH. Figure S2. Map of the plasmids pYLEX1, pYLEX1-2 and pYLEX1-3. Figure S3. Map of the plasmid pYLEX1-A1HB. Figure S4. Map of the plasmid pYLEX1-A2CR. Figure S5. Map of the plasmid pYLEX1-A2CREuET. Figure S6. Restriction map of pYLEX1-NEW. Plasmid pYLEX1-NEW with an improved version of MCS was constructed by adding XbaI, AvrII, MfeI and MluI sites into the BamHI site of pYLEX1. Plasmid pYLEX1-NEW contains two BamHI sites. Details on DNA sequence alignment and additional Materials and Methods are also provided as additional information.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-γ-linolenic acid (20:3 Δ(8,11,14)), resulting in arachidonic acid (20:4 Δ(5,8,11,14)) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5'-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5'-upstream region of other delta5 desaturases showed that the 5'-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5'-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5'-upstream region of fad5 could drive the expression of green fluorescent protein.

In order to investigate the regulatory mechanisms of Mortierella alpina desaturase genes by low temperature and exogenous unsaturated fatty acids (UFAs) at the transcriptional level.We performed time-course studies of fatty acid desaturase gene expression by real-time PCR and determined fatty acid desaturase gene promoter activity using promoter-reporter constructs.Relative expression results in real-time PCR showed that the mRNA levels of three fatty acid desaturase genes (Fad6, Fad12 and Fad3) were rapidly and transiently enhanced after 1 h of shifting to low temperature, in contrast, high concentration of exogenous oleic acid (OA) which was a monounsaturated fatty acid containing a D9 double bond suppressed the transcription of these genes and the transcriptional response appeared to be rapid and transient. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. The pFAD6 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by addition of UFAs to the media, and alpha-linolenic acid (ALA) containing three double bonds appeared to have a more effective inhibition than linoleic acid (LA) and OA.These results indicate that there may be post-transcriptional control and other modes of regulation of UFAs synthesis in M. alpina when facing different stimuli such as low temperature and exogenous unsaturated fatty acids besides the regulation in the transcription of fatty acid desaturase genes at the initial stage. Also, there may be an unknown end-product (changes in fatty acid compositions) feedback regulation in the transcription of fatty acid desaturase genes to maintain cellular UFAs' homeostasis. In a word, we assessed mechanisms of transcriptional regulation in M. alpina fatty acid desaturase gene expression for the first time and we wish to make it possible to obtain a better understanding of the mechanisms and get some theoretical knowledge to offer some guidance to the industrial production of UFAs by transgenic technology and microbial fermentation technology.

The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway.

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆(12)-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆(15)-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆(9)-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Here we investigated the regulation of Pichia pastoris desaturase genes by low temperature and exogenous fatty acids in the late-exponential phase at the transcriptional level. Time-course studies of gene expression showed that mRNA levels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous oleic acid. Stearic acid showed no obvious repression of mRNA levels of Fad12 and Fad15 and a slight increase in Fad9A and Fad9B mRNA levels. Using a promoter–reporter gene construct, we demonstrated that the pFAD15 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by unsaturated fatty acids. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. Disruption of Spt23 resulted in a decrease in transcript levels of Fad9A and Fad9B, but had little effect on the other desaturase genes. Consistent with these observations, a decrease in the relative amount of oleic acid (OLA) and an increase in the relative content of linoleic acid and ALA with different degrees were clearly observed in the stationary phase cells of ΔSpt23 mutant. Further analysis showed that the effect of low-temperature activation and OLA inhibition on expression of Fad9A and Fad9B seemed to disappear after disruption of the Spt23 gene, which indicated that Spt23p is essential for the expression of two Δ9-desaturase genes internally and probably involved in the regulation of Δ9-desaturase genes transcription in response to external stimuli, and thereby plays a role in the synthesis of OLA.

Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-∆4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ∆4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomyces cerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ∆4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-∆4-PUFAs-specific desaturase activity.

On the molecular and biochemical levels, the effects of different carbon sources on biomass production, fatty acid biosynthesis, and gene expression of three desaturases were investigated in Mortierella alpina ATCC 16266, at a stationary phase, which is an important filamentous fungus capable of producing various polyunsaturated fatty acids (PUFAs). The maximum mycelial biomass was achieved using sucrose as carbon source. However, the highest productivity of total lipids was shown to be no biomass associated. In addition, glucose was the preferred carbon source for the expression of three desaturase genes compared to others, but the change at the corresponding fatty acid product's level of these desaturase genes was not in accordance with the change measured at the mRNA level among those carbon sources that we utilized. Significant discrepancies between the mRNA expression and the product abundance may indicate post-transcriptional regulatory mechanisms of these desaturases.

Background Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical. Results We engineered Saccharomyces cerevisiae to produce fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences. To boost the ability of S. cerevisiae to produce the aforementioned esters, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted to increase flux towards fatty acyl-CoAs. In addition, five isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, and Adh7) targeted into the mitochondria were overexpressed to enhance production of alcohol precursors. By combining these engineering strategies with high-cell-density fermentation, over 230 mg/L fatty acid short- and branched-chain alkyl esters were produced, which is the highest titer reported in yeast to date. Conclusions In this work, we engineered the metabolism of S. cerevisiae to produce biodiesels in the form of fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters. To our knowledge, this is the first report of the production of fatty acid isobutyl and active amyl esters in S. cerevisiae. Our findings will be useful for engineering S. cerevisiae strains toward high-level and sustainable biodiesel production. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0361-5) contains supplementary material, which is available to authorized users.