Deciphering and engineering high-light tolerant cyanobacteria for efficient photosynthetic cell factories

Development and utilization of“liquid sunshine”could be one of key solutions to deal with the issues of fossil fuel depletion and increasing carbon dioxide.Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis,and their activity accounts for~25%of the total carbon fixation on earth.More importantly,besides their traditional roles as primary producers,cyanobacteria could be modified as“photosynthetic cell factories”to produce renewable fuels and chemicals directly from CO_(2) driven by solar energy,with the aid of cutting-edging synthetic biology technology.Towards their large-scale biotechnological application in the future,many challenges still need to be properly addressed,among which is cyanobacterial cell factories inevitably suffer from high light(HL)stress during large-scale outdoor cultivation,resulting in photodamage and even cell death,limiting their productivity.In this review,we critically summarized recent progress on deciphering molecular mechanisms to HL and developing HL-tolerant chassis in cyanobacteria,aiming at facilitating construction of HLresistant chassis and promote the future application of the large-scale outdoor cultivation of cyanobacterial cell factories.Finally,the future directions on cyanobacterial chassis engineering were discussed.

Constructing efficient bacterial cell factories to enable one-carbon utilization based on quantitative biology:A review

Developing methylotrophic cell factories that can efficiently catalyze organic one-carbon(C1)feedstocks derived from electrocatalytic reduction of carbon dioxide into bio-based chemicals and biofuels is of strategic significance for building a carbon-neutral,sustainable economic and industrial system.With the rapid advancement of RNA sequencing technology and mass spectrometer analysis,researchers have used these quantitative microbiology methods extensively,especially isotope-based metabolic flux analysis,to study the metabolic processes initiating from C1 feedstocks in natural C1-utilizing bacteria and synthetic C1 bacteria.This paper reviews the use of advanced quantitative analysis in recent years to understand the metabolic network and basic principles in the metabolism of natural C1-utilizing bacteria grown on methane,methanol,or formate.The acquired knowledge serves as a guide to rewire the central methylotrophic metabolism of natural C1-utilizing bacteria to improve the carbon conversion efficiency,and to engineer non-C1-utilizing bacteria into synthetic strains that can use C1 feedstocks as the sole carbon and energy source.These progresses ultimately enhance the design and construction of highly efficient C1-based cell factories to synthesize diverse high value-added products.The integration of quantitative biology and synthetic biology will advance the iterative cycle of understand–design–build–testing–learning to enhance C1-based biomanufacturing in the future.

Design and construction of microbial cell factories based on systems biology

Environmental sustainability is an increasingly important issue in industry.As an environmentally friendly and sustainable way,constructing microbial cell factories to produce all kinds of valuable products has attracted more and more attention.In the process of constructing microbial cell factories,systems biology plays a crucial role.This review summarizes the recent applications of systems biology in the design and construction of microbial cell factories from four perspectives,including functional genes/enzymes discovery,bottleneck pathways identification,strains tolerance improvement and design and construction of synthetic microbial consortia.Systems biology tools can be employed to identify functional genes/enzymes involved in the biosynthetic pathways of products.These discovered genes are introduced into appropriate chassis strains to build engineering microorganisms capable of producing products.Subsequently,systems biology tools are used to identify bottleneck pathways,improve strains tolerance and guide design and construction of synthetic microbial consortia,resulting in increasing the yield of engineered strains and constructing microbial cell factories successfully.

Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies

The global market demand for natural astaxanthin is rapidly increasing owing to its safety,the potential health benefits,and the diverse applications in food and pharmaceutical industries.The major native producers of natural astaxanthin on industrial scale are the alga Haematococcus pluvialis and the yeast Xanthopyllomyces dendrorhous.However,the natural production via these native producers is facing challenges of limited yield and high cost of cultivation and extraction.Alternatively,astaxanthin production via metabolically engineered non-native microbial cell factories such as Escherichia coli,Saccharomyces cerevisiae and Yarrowia lipolytica is another promising strategy to overcome these limitations.In this review we summarize the recent scientific and biotechnological progresses on astaxanthin biosynthetic pathways,transcriptional regulations,the interrelation with lipid metabolism,engineering strategies as well as fermentation process control in major native and non-native astaxanthin producers.These progresses illuminate the prospects of producing astaxanthin by microbial cell factories on industrial scale.

Development of fungal cell factories for the production of secondary metabolites:Linking genomics and metabolism

The genomic era has revolutionized research on secondary metabolites and bioinformatics methods have in recent years revived the antibiotic discovery process after decades with only few new active molecules being identified.New computational tools are driven by genomics and metabolomics analysis,and enables rapid identification of novel secondary metabolites.To translate this increased discovery rate into industrial exploitation,it is necessary to integrate secondary metabolite pathways in the metabolic engineering process.In this review,we will describe the novel advances in discovery of secondary metabolites produced by filamentous fungi,highlight the utilization of genome-scale metabolic models(GEMs)in the design of fungal cell factories for the production of secondary metabolites and review strategies for optimizing secondary metabolite production through the construction of high yielding platform cell factories.

Functional expression and regulation of eukaryotic cytochrome P450 enzymes in surrogate microbial cell factories

Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions,synthesis of diverse metabolites,detoxification of xenobiotics,and con-tribution to environmental adaptation.Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological,pharmaceutical and chemical industry applica-tions.Nevertheless,the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range.This review is focused on comprehen-sive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories,aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future.We also highlight the functional significance of the previously underrated cytochrome P450 reductases(CPRs)and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.

Efficient production of chemicals from microorganism by metabolic engineering and synthetic biology

The use of traditional chemical catalysis to produce chemicals has a series of drawbacks,such as high dependence on fossil resources,high energy consumption,and environmental pollution.With the development of synthetic biology and metabolic engineering,the use of renewable biomass raw materials for chemicals synthesis by constructing efficient microbial cell factories is a green way to replace traditional chemical catalysis and traditional microbial fermentation.This review mainly summarizes several types of bulk chemicals and high value-added chemicals using metabolic engineering and synthetic biology strategies to achieve efficient microbial production.In addition,this review also summarizes several strategies for effectively regulating microbial cell metabolism.These strategies can achieve the coupling balance of material and energy by regulating intracellular material metabolism or energy metabolism,and promote the efficient production of target chemicals by microorganisms.

In silico cell factory design driven by comprehensive genome‑scale metabolic models:development and challenges

Genome-scale metabolic models(GEMs)have been widely used to design cell factories in silico.However,initial flux balance analysis only considers stoichiometry and reaction direction constraints,so it cannot accurately describe the distribution of metabolic flux under the control of various regulatory mechanisms.In the recent years,by introducing enzymology,thermodynamics,and other multiomics-based constraints into GEMs,the metabolic state of cells under different conditions was more accurately simulated and a series of algorithms have been presented for microbial phenotypic analysis.Herein,the development of multiconstrained GEMs was reviewed by taking the constraints of enzyme kinetics,thermodynamics,and transcriptional regulatory mechanisms as examples.This review focused on introducing and summarizing GEMs application tools and cases in cell factory design.The challenges and prospects of GEMs development were also discussed.

Systematic optimization of the yeast cell factory for sustainable and high efficiency production of bioactive ginsenoside compound K

Ginsenoside Compound K(CK)has been recognized as a major functional component that is absorbed into the systemic circulation after oral administration of ginseng.CK demonstrates diverse bioactivities.A phase I clinical study indicated that CK was a potential candidate for arthritis therapy.However,a phase II clinical study was suspended because of the high cost associated with the present CK manufacturing approach,which is based on the traditional planting-extracting-biotransforming process.We previously elucidated the complete CK biosynthetic pathway and realized for the first time de novo biosynthesis of CK from glucose by engineered yeast.However,CK production was not sufficient for industrial application.Here,we systematically engineered Saccharomyces cerevisiae to achieve high titer production of CK from glucose using a previously constructed protopanaxadiol(PPD)-producing chassis,optimizing UGTPg1 expression,improving UDP-glucose biosynthesis,and tuning down UDP-glucose consumption.Our final engineered yeast strain produced CK with a titer of 5.74 g/L in fed-batch fermentation,which represents the highest CK production in microbes reported to date.Once scaled-up,this high titer de novo microbial biosynthesis platform will enable a robust and stable supply of CK,thus facilitating study and medical application of CK.

Synthetic biology for sustainable food ingredients production:recent trends

Problems with food security result from increased population,global warming,and decrease in cultivable land.With the advancements in synthetic biology,microbial synthesis of food is considered to be an efficient alternate approach that could permit quick food biosynthesis in an eco-friendly method.Furthermore,synthetic biology can be assumed to the synthesis of healthy or specially designed food components like proteins,lipids,amino acids and vitamins and widen the consumption of feedstocks,thus offering possible resolutions to high-quality food synthesis.This review describes the impact of synthetic biology for the microbial synthesis of various food ingredients production.

Recent advances in screening amino acid overproducers

Microbial fermentation has contributed to 80%of global amino acid production.The key to microbial fermentation is to obtain fermentation strains with high performance to produce target amino acids with a high yield.These strains are primarily derived from screening enormous mutant libraries.Therefore,a high-throughput,rapid,accurate,and universal screening strategy for amino acid overproducers has become a guarantee for ob-taining optional amino acid overproducers.In recent years,the rapid development of various novel screening strategies has been witnessed.However,proper analysis and discussion of these innovative technologies are lacking.Here we systematically reviewed recent advances in screening strategies:the auxotrophic-based strategy,the biosensor-based strategy,and the latest translation-based screening strategy.The design principle,application scope,working efficiency,screening accuracy,and universality of these strategies were discussed in detail.The potential for screening nonstandard amino acid overproducers was also analyzed.Guidance for the improvement of future screening strategies is provided in this review,which could expedite the reconstruction of amino acid overproducers and help promote the fermentation industry to reduce cost,increase yield,and improve quality.

Advances in microbial engineering for the production of value‑added products in a biorefinery

Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process.Thus,improving microbial capacity to maximize product yield,productivity,and titer has been recognized as a central goal for bioengineers and biochemists.The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production.The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways.New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity.In this review,the recent advances in building efficient biorefineries were showcased.In addition,the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.

Engineering yeast for bio-production of food ingredients

The provision of an adequate and high-quality food supply is a challenging issue due to the continued growth of the population and the reduction of arable land resources.To solve these problems,new and efficient food manufacturing processes need to be developed to meet the needs for healthy and nutritious food.Metabolic engineering of microorganisms is a feasible approach to alleviate this problem due to its efficient biosynthesis.For thousands of years,yeast has been used as a cell factory for manufacturing bread,beer and wine.And the development of synthetic biology expands its ability for synthesis of food ingredients,fuels,pharmaceuticals and chemical products.This mini review focuses on metabolic engineering of yeast cell factories to synthesize compounds that have been used as food ingredients with highlighting four food flavors.

Raising the production of phloretin by alleviation of by-product of chalcone synthase in the engineered yeast

Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.

Metabolic engineering using acetate as a promising building block for the production of bio‐based chemicals

The production of biofuels and biochemicals derived from microbial fermentation has received a lot of attention and interest in light of concerns about the depletion of fossil fuel resources and climatic degeneration.However,the economic viability of feedstocks for biological conversion remains a barrier,urging researchers to develop renewable and sustainable low-cost carbon sources for future bioindustries.Owing to the numerous advantages,acetate has been regarded as a promising feedstock targeting the production of acetyl-CoA-derived chemicals.This review aims to highlight the potential of acetate as a building block in industrial biotechnology for the production of bio-based chemicals with metabolic engineering.Different alternative approaches and routes com-prised of lignocellulosic biomass,waste streams,and C1 gas for acetate generation are briefly described and evaluated.Then,a thorough explanation of the metabolic pathway for biotechnological acetate conversion,cel-lular transport,and toxin tolerance is described.Particularly,current developments in metabolic engineering of the manufacture of biochemicals from acetate are summarized in detail,with various microbial cell factories and strategies proposed to improve acetate assimilation and enhance product formation.Challenges and future development for acetate generation and assimilation as well as chemicals production from acetate is eventually shown.This review provides an overview of the current status of acetate utilization and proves the great potential of acetate with metabolic engineering in industrial biotechnology.

A combined strategy for the overproduction of complex ergot alkaloid agroclavine

Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin Ⅲ to baccatin Ⅲ

10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor.Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell.Then,we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatinⅢ.We found that baccatinⅢcould be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+under alkaline condition.Thus,we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters reached 2,3 and 4.6 g/L,respectively,in a 3-L bioreactor culture when 2,3 and 6 g/L of 10-DAB were supplied.Our study provides an environmentfriendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis.The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol

Methylotrophic yeasts and bacteria, which can use methanol as carbon and energy source, have beenwildly used as microbial cell factories for biomanufacturing. Due to their robustness in industrial harshconditions, methylotrophic yeasts such as Pichia pastoris have been explored as a cell factory forproduction of proteins and high-value chemicals. Methanol utilization pathway （MUT） is highlyregulated for efficient methanol utilization, and the downstream pathways need extensively constructedand optimized toward target metabolite biosynthesis. Here, we present an overview of methanolmetabolism and regulation in methylotrophic yeasts, among which we focus on the regulation of keygenes involved in methanol metabolism. Besides, the recent progresses in construction and optimizationof downstream biosynthetic pathways for production of high value chemicals, such as polyketides, fattyacids and isoprenoids, are further summarized. Finally, we discuss the current challenges and feasiblestrategies toward constructing efficient methylotrophic cell factories may promote wide applications inthe future.

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity.Traditional approaches for producing them rely on direct extraction from Panax ginseng,enzymatic catalysis or chemical synthesis,all of which result in low yield and high cost.Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products.Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-OGlc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system.The titers of F2 and 3β,20S-Di-O-GlcDM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level,respectively,by multistep metabolic engineering strategies.Additionally,pharmacological evaluation showed that both F2and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-GlcDM was even better.Furthermore,the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor.To our knowledge,this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM,and achieving their de novo biosynthesis by the engineered yeasts.Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass,which lays a foundation for drug research and development.

Engineering Saccharomyces cerevisiae for efficient production of recombinant proteins

Saccharomyces cerevisiae is an excellent microbial cell factory for producing valuable recombinant proteins because of its fast growth rate,robustness,biosafety,ease of operability via mature genomic modification technologies,and the presence of a conserved post-translational modification pathway among eukaryotic organisms.However,meeting industrial and market requirements with the current low microbial production of recombinant proteins can be challenging.To address this issue,numerous efforts have been made to enhance the ability of yeast cell factories to efficiently produce proteins.In this review,we provide an overview of recent advances in S.cerevisiae engineering to improve recombinant protein production.This review focuses on the strategies that enhance protein production by regulating transcription through promoter engineering,codon optimization,and expression system optimization.Additionally,we describe modifications to the secretory pathway,including engineered protein translocation,protein folding,glycosylation modification,and vesicle trafficking.Furthermore,we discuss global metabolic pathway optimization and other relevant strategies,such as the disruption of protein degradation,cell wall engineering,and random mutagenesis.Finally,we provide an outlook on the developmental trends in this field,offering insights into future directions for improving recombinant protein production in S.cerevisiae.