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A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

ABSTRACT: Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10?g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

SUBMITTER: Vickery CR

PROVIDER: S-EPMC6701952 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Vickery Christopher R CR Cardenas Javier J Bowman Marianne E ME Burkart Michael D MD Da Silva Nancy A NA Noel Joseph P JP

Biotechnology and bioengineering 20180313 6

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site varia ...[more]

PMID: 29457628

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Project description:BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.ConclusionsSaccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.

Project description:Saccharomyces cerevisiae is bradytroph for class B vitamins, it means that yeast cells exhibit slower growth in the absence of an external source of these metabolites. Alleviating these nutritional requirements for optimal growth performance would represent a valuable phenotypic characteristic for industrial strains since this would result in cheaper processes that would also be less susceptible to contaminations. In the present study, suboptimal growth of S. cerevisiae in absence of either pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and biotin were corrected by single or double gene overexpression of native FMS1, ABZ1/ABZ2, SNZ1/SNO1, INO1 and the Cyberlindnera fabianii BIO1, respectively. Several strategies were attempted to improve growth of S. cerevisiae CEN.PK113-7D in absence of thiamine, revealing that overexpression of THI4 and THI4/THI5 was able to improve growth up to 83% of the maximum specific growth rate of the reference CEN.PK113-7D in medium including all vitamins. Although the initial aim of this study was to combine all identified mutations in a single strain, the engineered strain IMX2210 only harboured genes to correct biotin, pABA, pantothenate and inositol bradotrophies. Firstly, this strain was fast-growing at a maximum specific growth rate of 0.28 ± 0.01 h-1 in medium devoid of all vitamins. Secondly, this strain exhibited physiological variables in aerobic glucose limited chemostat cultures at a dilution rate of 0.1 h-1 in absence of vitamins similar to that of the reference strain CEN.PK113-7D grown in the same conditions but in a fully supplemented complete medium. These physiological similarities were further emphasized by the limited differences observed in comparative transcriptome analysis from the chemostat culture grown cells that were essentially affecting genes of the class B vitamins biosynthetic pathways. This work paves the way towards construction of the first fast growing vitamin-independent S. cerevisiae strain.

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A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Publications

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

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