Impact of oxygen supply on production of terpenoids by microorganisms:State of the art

Ting-Ting Liu,Han Xiao,Jian-Hui Xiao,Jian-Jiang Zhong,*

1 State Key Laboratory of Microbial Metabolism,Joint International Research Laboratory of Metabolic and Developmental Sciences,Laboratory of Molecular Biochemical Engineering and Advanced Fermentation Technology,Department of Bioengineering,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China

2 Zunyi Municipal Key Laboratory of Medicinal Biotechnology,Center for Translational Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China

ABSTRACT Terpenoids are a class of high value-added natural products with a variety of biological functions.Genetically engineered microorganisms,such as those of Escherichia coli and Saccharomyces cerevisiae,have merits in producing plant or fungus-derived terpenoids,due to their mature genetic manipulation,simple nutrient demand and fast growth.Oxygen,as a key environmental factor,is particularly important to microbial metabolism and growth,and suitable oxygen supply is viewed as a prerequisite for realizing highly efficient production of terpenoids by engineered microorganisms.In this article,the role of oxygen in regulating terpenoid bioproduction is overviewed from the viewpoints of cellular carbon metabolism,energy metabolism and terpenoid anabolism.Strategies on adjusting oxygen availability to microorganisms,including genetic modification of cellular metabolism related with oxygen utilization,are summarized and discussed,to provide helpful information for further improvement of terpenoid biosynthesis by microbes.

Keywords:Terpenoid Microorganisms Oxygen supply Chassis modification

1.Introduction

Terpenoids,also often called as isoprenoids,belong to a family of natural products with a remarkable diversity in chemical structures.Diverse numbers of isoprene (C5H8) are combined to form monoterpenoids,diterpenoids,triterpenoids,tetraterpenoids and polyterpenoids (Fig.1) [1].The complexity of chemical structure endows terpenoids with abundant biological and pharmacological activities.Owing to their important functions as pollination attractants,repellents,antifeedants and pathogen-inducible phytoalexins,terpenoids also play a key role in ecosystems [2].A wide range of bioactivities against cancer,malaria and inflammation establish the important status of terpenoids in human disease therapy and prevention [3].

In general,natural terpenoid producers are plants and fungi,which possess vast quantities of terpenoid synthases,including prenylelongases and terpene cyclases [4].But,due to their long growth cycle under the natural environment,low content based on cell weight and inefficient extraction,their popularity has been compromised by the difficulty of producing terpenoids on large sacle and cheaply.Microbial cell factory is looked at as an effective approach for large-scale production of terpenoids,because of their mature genetic manipulation,simple nutrient demand and short growth circle.The mevalonate (MVA) and methylerythritol phosphate (MEP) pathways,two independent pathways to synthesize terpenoid precursors,exist widely in eukaryotes,archaea [5,6]and multiple bacteria[7],which facilitate the genetic modification for heterogeneous microbial synthesis of terpenoids.Until now,microorganisms have been well applied to the biosynthesis of terpenoids such as santalene [8],nootkatone[9],carotenoid [10]and ganodericacid3-hydroxy-lanosta-8,24-dien-26oicacid(GA-HLDOA) [11].Here,three examples of microbial cell factory for terpenoid production on (semi-)industrial scales are given.Astaxanthin is naturally present in various marine animals and microorganisms.By the means of illuminated fermentation and glucose feeding strategy,Xanthophyllomyces dendrorhous gave a yield of 350 mg·L-1at the 800-L scale,revealing an improved semi-industrial process for astaxanthin biosynthesis [12].Lutein is known as a carotenoid with its function in protecting eyes.As the cultivation was scaled up to 240,000 L,the yield of lutein reached 263.13 mg·L-1in Chlorella vulgaris,providing a solid basis for large-scale production of lutein [13].Artemisinin,a valuable drug against malaria,naturally occurs in minute amounts in Artemisia species with a very low yield in the range of 0.01%–0.8%(by dry mass).The industrial process combining fermentation of engineered Saccharomyces cerevisiae (baker’s yeast) for highyielding biological production of artemisinic acid (a precursor of artemisinin) with a photochemical process for the conversion of artemisinic acid to artemisinin at Sanofi has already produced tons of artemisinin [14].

Fig.1.Terpenoid biosynthesis pathway designed in microorganisms.The MVA and MEP pathways and TCA cycle are indicated in blue,gray and green frames,respectively.The red box means a series of modifications of terpenoid core skeletons including the catalytic reactions of cytochrome P450s.Abbreviations:AKG,α-ketoglutaric acid;CDPME,4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol;CDP-MEP,CDP-ME 2-phosphate;DMAPP,dimethylallyl diphosphate;DXP,1-deoxy-D-xylulose 5-phosphate;FPP,farnesyl diphosphate;GPP,geranyl diphosphate;G3P,glyceraldehyde 3-phosphate;Glucose-6P,glucose-6-phosphate;GGPP,geranylgeranyl diphosphate;HMG-CoA,3-hydroxy-3-methylglutaryl CoA;HMBPP,1-hydroxy-2-methyl-2-butenyl 4-diphosphate;IPP,isopentenyl diphosphate;IP,isopentenyl phosphate;MVP,5-phosphomevalonate;MVPP,5-diphosphomevalonate;MEcPP,2-C-methyl-D-erythritol 2,4-cyclodiphosphate;MEP,2-C-methyl-D-erythritol 4-phosphatel;OAA,oxaloacetic acid;PPP,pentose phosphate pathway.

Oxygen,a key environmental factor,is crucial for most of engineered microorganisms used for terpenoid production,which serves as a substrate or an electron acceptor to satisfy the need of aerobic growth and metabolite synthesis[15].During terpenoid synthesis,pyruvate,glyceraldehyde 3-phosphate(G3P)and acetylcoA,the precursors of the MEP and the MVA pathways,are synthesized by central carbon metabolism such as glycolysis.The next step is just converting these precursors into different downstream metabolites,in which oxygen plays a decisive role for the fate of precursors.By responding to oxygen levels,cell metabolic modes can transform between respiration and fermentation,resulting in the change of metabolic flux and secondary metabolite biosynthesis.The synthesis of terpenoids from core skeletons undergoes diverse post-modifications,in which cytochrome P450s (CYPs)play a key role [16].CYPs are monooxygenases which introduce one oxygen atom into the substrate and contribute to the oxyfunctionalization of multiple compounds with the involvement of oxygen [17].Enhancing oxygen delivery is favorable to oxygenase activity [18].Heme,known as prosthetic group of cytochromes,was demonstrated to have relevance to dissolved oxygen (DO) as well [19].Enzymes involved in the formation of heme,such as coproporphyrinogen III oxidase and protoporphyrinogen IX oxidase,are oxygen-dependent,suggesting that DO levels might affect the catalysis efficiency of CYPs by regulating cellular heme concentrations.Besides,oxygen serves as the preferred terminal electron acceptor in respiratory chain to ensure a sufficient supply of ATP for cell growth and secondary metabolism [20].Therefore,oxygen supply is a critical factor in the synthesis of terpenoids.However,improper oxygen levels may exert a negative effect on cell physiological state.For example,hypoxia was shown to cause negative changes in terpenoid biosynthesis,TCA cycle and amino acid metabolism inAspergillus fumigatus[21].Excess oxygen had disadvantages including DNA injury,enzyme deactivation,membrane breakage and lipid peroxidation through the generation of excessive reactive oxygen species (ROS),seriously affecting cell growth and metabolism [22].

As a result,suitable oxygen level to maintain the balance of oxygen supply and cellular requirement is vital to achieve mass production of terpenoids.This review aims to summarize various strategies used for efficient synthesis of terpenoids by using bioprocess engineering and genetic modification to guarantee oxygen availability to cells.Moreover,comprehensive analysis and indepth discussion are put on the role of oxygen in terpenoid biosynthesis,which offer rational adjustment of oxygen supply.

2.Manipulation of Oxygen Supply for Terpenoid Production by Changing Cultivation Conditions

During cell culture,oxygen availability significantly impacts cell growth and metabolism of most aerobic microorganisms.Hence,ensuring that oxygen supply in fermentation is sufficient to meet the demand of production strains is important to achieve super productivity and low production cost.Researches have been attempted to control cultivation conditions to balance the oxygen supply and cellular oxygen uptake,which provide opportunities for effective terpenoid accumulation.Some typical examples are summarized in Table 1.

2.1.Manipulation of agitation and aeration

High cell density is a prerequisite to realize high-yield and highefficiency production of secondary metabolites,in which suitable oxygen is required.Affected by nutrients and salt ions,oxygen solubility is generally lower in microbial medium than in water.The aim of increasing DO to acquire high biomass levels is generally realized by manipulating agitation and aeration [42].The oxygen transfer process may be closely linked to shaking speed and liquid volume in flasks,as well as impeller tip speed and aeration rate in fermentation reactors.In shake-flask cultures,the oxygen transfer coefficient (KLa) showed a strong correlation with shaking speed and liquid volume,and an obvious increase in carotenoid yield was achieved by setting high speed and low liquid volume [31].In a stirred tank reactor at an aeration rate of 1.5 vvm,maximum β-carotene yield(1.5 kg·m-3)was obtained at a low impeller speed(150 r·min-1),while the highest β-carotene productivity(0.08 kg·m-3·d-1) was achieved at a higher impeller speed of 500 r·min-1[43].High agitation speed and aeration rate(300 r·min-1,1000 ml·min-1) enhanced the yield of astaxanthin and the ratio of astaxanthin in total carotenoid accumulation[44].Furthermore,DO-triggered pulse fed-batch was applied to increase triterpenoid accumulation,in which DO level was maintained above 30% using a stirring cascade [45].Respiratory quotient (RQ)-controlled feed strategy,which correlates RQ with feed rate to improve cell density and reduce ethanol accumulation,has been successfully used for high production of terpenoid such as α-santalene,farnesene and santalene [8,24].Among the above strategies to promote oxygen availability,excessive shearing forces caused by agitation would result in damage to microbes,while supplying oxygen-enriched air may be too expensive.Increasing air pressure is known as an effective alternative method to enhance DO by improving oxygen mass transfer,leading to the enhancement of cell density and product yield [46].Due to the merit of low industrial cost,pressurization has been widely used in a series of aerobic microbial fermentation processes,such as in large-scale production of β-xylosidase byPichia pastoris[47],and this pressurization strategy may be also applied to terpenoid fermentation by engineered microbes.

Through omics analysis,microbes were proved to switch metabolic phases between aerobic fermentation and respiration by metabolic flux rearrangement during whole lifespan,implying fluctuant oxygen requirements of cells at different growth stages[48].Therefore,ensuring oxygen supply at the optimal concentration in different growth stages during cultivation process is crucial to high production of terpenoids.For example,a twostage cultivation strategy replaced a constant DO strategy to deal with the tradeoff between the accumulation of biomass and metabolites to satisfy their different needs of oxygen.The highest ganoderic acid production ((976 ± 35) mg·L-1) and cell dry mass ((20.8 ± 0.1) g·L-1) were obtained by two-stage oxygen supply method inGanoderma lucidum[49].A similar strategy was applied to triterpenoid production inGanoderma lucidumACCC G0119,leading to a higher yield in that process [29].In addition,a multi-stage oxygen control strategy was reported for efficient production of microbial oil inMortierella alpina[50],which might be applicable to terpenoid production in microbial fermentation.

Table 1 Representative examples of oxygen supply strategies applied to terpenoid production

2.2.Addition of oxygen vectors

Maintaining a high oxygen supply by regulating fermentation parameters may bring negative impacts to cell culture such as strong shear force,foam formation or excessive power input,which are of side effect and not economical.One simple and efficient approach may be the addition of an oxygen vector to liquid medium,which is insoluble in water but dissolves approximately 15–20 times more oxygen than water[51].Addition of oxygen vectors leads to increased oxygen mass transfer rate and thus enhances DO levels.More importantly,oxygen vectors have very low or no toxicity to cells and can even be used as supplementary carbon and energy source to satisfy the need of cells,resulting in higher biomass concentrations [52].Extensive studies confirm the feasibility and effectiveness of this strategy in terpenoid production.n-Hexadecane,known as a common oxygen vector,was proved to improve cell growth and carotenoid yield effectively.Whenn-hexadecane concentration in the medium accounted for 9.0%(v/v),the yield of biomass and carotenoid showed an increase of 13.0% and 57.6%,respectively [32].Crude soybean oil has been successfully applied in lycopene production byBlakeslea tribalism.As a low-cost carbon source and an efficient oxygen vector,crude soybean oil stimulated the synthesis of lycopene and enhanced fermentation efficiency while reduced production cost [53].Furthermore,it is found that several oxygen vectors such asn-dodecane can be used as extractants to remove metabolites in situ.Several intermediate metabolites in terpenoid synthesis pathway such as dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate(IPP) are known to be toxic to cells,and excessive accumulation may adversely affect cell growth and metabolism [54].Addition of extractive phase is capable of overcoming the limitation of cellular storage space,relieving product feedback inhibition and weakening cytotoxicity,resulting in the improvement of terpenoid production [55].

On the other hand,the addition amount of oxygen vector should be taken carefully.For example,n-hexadecane at high volume fractions decreased oxygen transfer coefficient [32].The negative effect of highn-hexadecane level was probably due to a significant increase in the apparent viscosity of medium,which impeded oxygen transfer.The film formed by organic liquid on the gas–liquid surface was another obstacle to DO delivery.Therefore,suitable use of oxygen vector is important to maximize the fermentation efficiency.

3.Effect of Oxidative Stress on Terpenoid Biosynthesis under Various Oxygen Supply Conditions

As well known,ROS is unavoidable in aerobic culture systems.The imbalance between the generation and elimination of ROS could increase local ROS levels,resulting in the occurrence of cellular oxidative stress response [56].Both excessive oxygen and hypoxia act as switches to stimulate ROS formation[21].Microbes possess two systems to protect against oxidative damage.One is the enzymatic defense system which consists of various antioxidant enzymes,including catalase (CAT),superoxide dismutase(SOD),ascorbate peroxidase (APX) and glutathione peroxidase(GPX).The other is non-enzymatic defense system,in which multiple antioxidant compounds play a key role.A variety of terpenoids have been shown to possess antioxidant potentials for eliminating cellular ROS and alleviating the inhibition of cell growth,which include lycopene,lutein,perillic acid,eremanthin and ganoderic acids[57].For instance,Nanouet al.increased cellular ROS levels by enhancing aeration in cultivation ofBlakeslea trispora,and achieved a higher carotene content for the cells to cope with oxidative stress [34].Here,the biosynthesis of antioxidative terpenoids is considered relevant to oxidative stress,implying a new approach for improving terpenoid yield,i.e.,oxidative stress as an inducible factor for terpenoid biosynthesis.Research confirmed that oxidative stress was an effective strategy used for enhancing the accumulation of β-carotene [58].The expression levels of key enzyme genes related to β-carotene biosynthesis were increased by the stimulation of oxidative stress.And the aerobic metabolism of glucose was significantly enhanced,showing the possibility of generating more ATP and NADPH.For the above reasons,β-carotene yields increased by nearly 38%.The positive regulative function of oxidative stress was applied to ganoderic acid production as well.Expression levels of major antioxidant enzymes(CAT,SOD,APX and GPX) responded to the change of cellular ROS levels [59].ROS was suggested to contribute to the synthesis of ganoderic acid by activating Ca2+signaling(Fig.2).But,ROS generated by nicotinamide adenine dinucleotide phosphate oxidases(Nox)appeared to have a positive correlation to cellular Ca2+levels[60];and in the GPx-silenced strain,Ca2+levels decreased with the increase of ROS concentrations [61],implying the complexity of the interaction between ROS and Ca2+,which needed further investigation.

In addition to its role as a stimulator of terpenoid synthesis,oxidative stress acts as an inhibitory factor to cell growth and metabolism.Excessive ROS triggers a certain degree of cell damages and even cell death.For instance,high aeration rates and the addition of butylated hydroxytoluene and H2O2resulted in a very high level of ROS inBlakeslea trispora,initiating cell autolysis and a decrease in biomass at the later growth stage.β-Carotene yield showed a significant decrease,indicating that cells may resist serious oxidative damage at the expense of antioxidant terpenoid levels to survive[58].Moreover,malondialdehyde,a cytotoxic substance derived from lipid peroxidation,achieved a higher concentration,implying the damage of ROS to cytomembrane inSaccharomyces cerevisiae[62].Therefore,the above facts indicate that there exists a critical value of ROS which determines whether the regulatory function of oxidative stress is positive or negative to cellular metabolism.

4.Regulation Mechanism of Oxygen on Terpenoid Biosynthesis

Multiple strategies based on controlling oxygen supply have been successfully used in terpenoid production,reflecting the importance of oxygen in terpenoid production process.The primary function of oxygen as a substrate is critical to cellular metabolisms of aerobic organisms for their substrate utilization and energy supply.Another role of oxygen in metabolism is its regulatory effect in a series of cell functions which respond to varying oxygen levels.Here,based on literature information,the regulation mechanism of oxygen is discussed from carbon metabolism,energy metabolism and terpenoid anabolism of cells as follows.

Fig.2.Schematic representation of ROS regulating ganoderic acid biosynthesis in Ganoderma lucidum.The dotted arrows indicate that regulatory mechanisms are still unclear,and the solid arrows indicate the known regulatory mechanisms.Abbreviations:APX,ascorbate peroxidase;CAT,catalase;GPX,glutathione peroxidase;GR,glutathione reductase;GSH,glutathione;GSSG,oxidized glutathion;NOX,dinucleotide phosphate oxidases;SOD,superoxide dismutase.

Central carbon metabolism:Pyruvate,G3P and acetyl-coA,crucial molecules in carbon metabolism,are known as the source of terpenoid precursors in the MVA pathway and the MEP pathway.Glycolysis,PPP,TCA cycle and oxidative phosphorylation affect the interconversion between NAD(P)+and their reduced form NAD(P)H,which influences CYPs enzyme activity and terpenoid synthesis through the regulation of coenzyme levels [63].As reported [64],cellular carbon metabolism can be reprogrammed in response to varying oxygen levels,leading to different metabolic operation modes.Metabolomic profiling of taxane production in response to different oxygen levels provides some information on the regulation mechanism of oxygen on cell metabolism[65].For example,under a low dissolved oxygen condition,cells exhibited a significant increase in the flux of glycolysis,supplying more substrates such as pyruvate for downstream metabolism;and most of pyruvate from glycolysis was used to produce acetyl-CoA for the synthesis of secondary metabolites,rather than flowing into the mitochondria to form ATP.The phenomenon was consistent with previous research [66],which implied that the flux of the TCA cycle was positively correlated with oxygen levels by regulating genes encoding TCA enzymes.Glycine,a key part of CYPs[67],showed a high metabolic flux at a low oxygen content which might contribute to P450 expression and taxane yield.Downregulated TCA cycle flux and adequate levels of precursors had a positive effect on the MVA pathway and generated more taxane skeletons.Furthermore,high oxygen levels were associated with more astaxanthin accumulation,implying that respiration mode may be more suitable for astaxanthin synthesis than fermentation[68].Improved NADPH availability might contribute to the enhanced astaxanthin yield.As reported [69],hyperoxic environment resulted in the enhancement of the PPP pathway and malic enzyme (ME) expression,which ensured a sufficient supply of NADPH under the high oxygen supply condition.

Energy metabolism:Fueling cellular activities with biochemical energy is crucial for microbial growth and secondary metabolite synthesis.There is a need of three ATPs in the MVA pathway to synthesize five-carbon building blocks,emphasizing the necessity of energy in terpenoid production [70].Oxygen,known as the preferred electron acceptor for energy metabolism,is able to determine the fate of energy metabolism,which can regulate the synthesis of secondary metabolites in view of energy metabolism.It was indicated that lycopene accumulation increased with the increase of oxygen levels,underlining the positive relevance between lycopene yields and oxygen supplies [71].In addition to the above effects of central carbon metabolism,energy metabolism provides a reasonable explanation from another perspective.As known,the synthesis of lycopene has a great need for energy(eight CTPs and eight ATPs per mole,respectively)[72].Enhanced cellular oxygen level led to higher protons generation and higher ATPase activity.The high proton flux could be used by ATPases to produce more ATP [73].Moreover,it was confirmed that slight changes in oxygen availability could result in multiple changes in the content and nature of terminal oxidases,affecting the composition of the respiratory chain and ATP synthesis [74].In microaerophilic environment,cytochromebdoxidase played a major role in transporting the electron to oxygen whereas cytochromebowas more active in aerobic condition[75].The expression of cytochromebooxidase increased with the increase of oxygen input,resulting in the enhanced respiration rate of cells for more ATP generation.Improved expression levels of key enzymes and higher proton flus satisfied the energy requirements for secondary metabolite synthesis under high oxygen condition.

Terpenoid anabolism:The expression and activity of enzymes in terpenoid biosynthesis pathway are directly related to terpenoid yields.Oxygen was observed to exert a regulatory effect on the expression of enzymes in MVA pathway [76].The maximal cell density ((29.8 ± 1.7) g·L-1) and the highest GA content ((1427.2 ±74.2) mg·L-1) were obtained at 80% oxygen.Compared with the control (air),the expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR),squalene synthase (SQS) and lanosterol synthase(LS)had a 1.7-,2.9-and 1.4-fold increase at 80%oxygen concentration,respectively.Aerobic conditions were also beneficial to gene expression levels of terpenoid anabolism in order to produce more carotenoid [77].Compared with anaerobic condition,the expression levels of carotenoid biosynthesis genes (crtMandcrtN)were improved by 5.3 and 5.9 times in aerobic conditions,respectively.At the same time,the MVA and isoprene biosynthesis pathways,known as typical pathways for carotenoid precursor formation,were affected by oxygen.The expression levels of HMGR and five major genes were improved by 1.2 to 2.3 times,revealing the potential of generating more precursors under aerobic condition.In addition to the regulatory function to gene expressions,a suitable oxygen level could stimulate oxygenase activities.CYP 1A1 and 1A2 amounts and their corresponding monooxygenase activities were increased by hyperoxia,providing a convincing evidence for oxygen regulating function [78].Furthermore,oxygen may indirectly affect enzyme activities by regulating cellular NAD(P)H and heme contents.As well known,several redox enzymes exist in terpenoid forming pathways,and their biochemical properties are NADPH or oxygen-dependent.HMGR,known as an important enzyme in the synthesis of terpenoids in heterologous hosts,is functional in the presence of NADPH,for instance[79].As mentioned,NAD(P)H contents change with DO levels by the regulation of oxygen on central carbon metabolism,leading to the regulation to enzyme catalysis.Heme,the prosthetic group of CYPs,requires the involvement of oxygen in the formation process.Cellular heme levels in response to different oxygen levels affect terpenoid yields by regulating enzyme activities [17].

5.Genetic Modification of Chassis Cells based on Oxygen Requirement

As a simple but effective approach,controlling oxygen supply via changing cultural conditions has achieved high yields of multiple terpenoids by affecting cell metabolism.However,the above strategies may increase production cost.Advancement in modern biotechnology makes it possible to optimize chassis cells by mining new microbe resources or modifying chassis cells to adapt for low oxygen conditions without the need for extra oxygen replenishment.

5.1.Use of terpenoid producers with low oxygen demand

In general,aerobic microorganisms act as the major producers of natural secondary metabolites,while the biosynthetic ability of anaerobic microorganisms is generally low.According to genomic sequence data,anaerobe is a rich storehouse of natural product biosynthetic gene clusters,showing the potential for terpenoid synthesis [80].Clostridium ljungdahliihas been actually proven as a heterologous host to synthesize mevalonate and isoprene in a more economical and environment-friendly way.Fructose and syngas (H2,CO2and CO) served as low-cost sources of carbon and reducing equivalents,and strictly anaerobic condition was required in whole process [81].Geobacter metallireducens,known as a strictly anaerobic bacterium,seems to possess several genes related with hopanoid formation,such as the genes for expressing two squalene-hopene cyclases.The occurrence of squalene and a series of hopanoids inG.metallireducensproved the ability of terpenoid synthesis in strictly anaerobic bacteria [82].However,insufficient practical experiences and immature genetic systems are barriers to the mass production of terpenoids in anaerobic host systems,in which further research is needed.

Cyanobacteria and microalgae,known as two typical photosynthetic microbes,are capable of generating isoprene and terpenoid hydrocarbons with sunlight,CO2and H2O as main energy/nutrition source,which have realized the biosynthesis of multiple terpenoids such as limonene,phellandrene,farnesene and amorphadiene [83].Oxygen derived from photosynthesis satisfied the need of cell respiratory and hydrogen produced by light reactions of photosynthesis was stored as terpenoid hydrocarbons [84].However,most of photosynthetic carbon was generally used to synthesize sugars in order to accumulate cell biomass,restricting the ability of terpenoid production in cyanobacteria and microalgae.The emergence ofBotryococcus brauniivarShowaoffers the possibility of redirecting more photosynthesis carbon into terpenoid biosynthesis pathway[85].The mechanism of natural high terpenoid accumulation is receiving attention,which is expected to be applied to more photosynthetic biological systems.

5.2.Modifying chassis cells to cope with low oxygen level

Manipulating microbial cells to keep optimal condition for cell growth and product synthesis with low demand for oxygen has drawn great attention recently,because of economic benefits and application potential.

Research was done to modify chassis for better cell metabolism under low oxygen conditions.One strategy was the use of microor anaerobic promoters,providing feasibility in stimulating product formation under low oxygen condition.For instance,anaerobically-inducednirBpromoter (PSnirB) anddmsApromoter(PdmsA)were applied in micro-aeration fermentation[86].Cascaded Pvgbrepeats (P8vgb) formed a novel promoter,resulting in stronger induction effect than single Pvgbunder microaerobic culture [87].Another strategy was to find gene targets for micro-or anaerobic metabolism regulation.RegB-RegA was proved to play an important role in many cellular processes by regulating anaerobic and aerobic expressions,including the synthesis of heme and cytochrome apoproteins related to terpenoids synthesis.The regulatory function of RegA was demonstrated to improve bacteriochlorophyll and carotenoid yields inRhodobacter capsulatusby upregulating gene expression [88].Several key global transcription factors such as fumarate nitrate reduction regulator (FNR) and anoxic redox control (ArcA) which serve as regulators for the anaerobic metabolism ofE.coli,may be applied as ideal modified targets [89].The reconstruction of novel anaerobic pathway instead of native pathway has been a systematic way to achieve targeted optimization of cells as well [90].However,above strategies are rarely reported in the field of terpenoid synthesis,which need further studies.

Introducing hemoglobin gene into chassis cells is conducive to improve the cellular capability of capturing oxygen.Three types of hemoglobins discovered in bacteria are single domain hemoglobin,flavohemoglobins(FlavoHb)and truncated hemoglobin(trHb),respectively.Vitreoscillahemoglobin gene(vgb),a typical model for single-domain hemoglobin,has been widely used as a powerful tool in the field of genetic modification to overcome oxygen deficiency and enhance value-added metabolite yields such as astaxanthin and carotenoids [91].Application of other bacteria hemoglobin was also reported,leading to the construction of various hemoglobin heterogeneous expression systems.Co-expression of trHb along with thecrtWZgenes encoding astaxanthin synthesis enzymes increased cellular oxygen availability and had the potential to improve the catalytic activity ofCrtWZenzymes,resulting in higher biomass and astaxanthin yields inMethylomonas[92].Novel hemoglobins generated by protein and gene engineering provide cellular oxygen availability with opportunities for further optimization.The construction of VHb mutants,single chain variants of VHb and chimeric proteins achieved optimal performance in heterologous hosts [93].

Moreover,lowering the need for oxygen through genetic methods is a targeted approach to provide more possibilities for further improving terpenoid synthesis in a hypoxic environment,which reduces the consumption of carbon from CO2emission and maximizes the shift of carbon to terpenoid synthesis pathway.By introducing four non-native metabolic reactions intoS.cerevisiaeto rearrange cellular carbon metabolism,the engineered strain could increase farnesene yield by 25%and decrease oxygen consumption by 75%,verifying the usability and superiority of metabolic rearrangement strategy [94].

6.Conclusions and Perspectives

Suitable oxygen supply is essential to cell growth and secondary metabolism in fermentation processes.Based on the literature analysis,regulation mechanisms of oxygen on terpenoid biosynthesis are summarized as follows:redirecting central metabolic pathway,regulating energy metabolism,and affecting gene transcriptions and functions in terpenoid anabolism.However,few reports have systematic analyses about the principles and associations of different regulatory mechanisms.Further study is required to explore the complex and crucial role of oxygen.Meanwhile,multiple functions of oxygen give us possibilities to establish an autoinducing oxygen-responsive switch to efficiently produce different compounds by a single strain,resulting in the creation of a productive process of time saving,cost reducing and quality guaranteeing[95].Now,a series of strategies to coordinate oxygen supply and demand provide opportunities for improving terpenoid production from multiple aspects such as fermentation engineering,genetic engineering and metabolic engineering.New bioreactor design serves as a powerful way to improve oxygen supply in cultivations as well [96].Continuous self-cycling fermentation was first used in lycopene production byS.cerevisiae,providing a new fermentation mode with cost efficiency and low pollution[97].With multi-level omics[98],abundant chassis cells with various characteristics will be analyzed and found,and more in-depth metabolic information will be revealed,which provide us with more selectivity in optimizing chassis cells.Fine-tuning host metabolism by various genome editing tools serves as a promising alternative for further improving terpenoid biosynthesis,which is a target-oriented direct approach to improve cellular properties.For instance,systematic manipulation of cell metabolism can maximize terpenoid synthesis as much as possible by reducing byproduct accumulation,minimizing metabolic loss of raw materials and redirecting the carbon flux to target products,which was successfully used in the highly efficient and low cost production of squalene and lycopene[99,100].In the future,strategies from bioengineering and synthetic biology perspectives can be performed with various combinations,which may lead to great advancement in developing terpenoid bioproduction organisms and processes[16,101].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key R&D Program of China (Nos.2019YFA0904800 and 2018YFA0901900) and the National Natural Science Foundation of China (No.31770037).The authors also appreciate Prof.Yan Sun for his kind invitation to this special issue.