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Improvement of betanin biosynthesis in Saccharomyces cerevisiae by metabolic engineering.

ABSTRACT: Betanin is a member of natural pigment betacyanins family and has extensive application in the food industry as an important natural red food colorant. Its relatively inefficient production in nature however hampers access to this phytochemicals through traditional crop-based manufacturing. Microbial bioproduction therefore represents an attractive alternative. Here, we present the construction of a Saccharomyces cerevisiae strain for betanin production. Through minimizing metabolic crosstalk, screening and modifying biosynthetic enzymes, enhancing pathway flux and optimizing fermentation conditions, a final titer of betanin of 28.7 mg/L was achieved from glucose at 25 °C in baffled shake-flask, which is the highest reported titer produced by yeast to our knowledge. This work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added betanin and other betacyanins.

SUBMITTER: Zhang L

PROVIDER: S-EPMC9672881 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Improvement of betanin biosynthesis in <i>Saccharomyces cerevisiae</i> by metabolic engineering.

Zhang Lijuan L Liu Xue X Li Jiawei J Meng Yonghui Y Zhao Guang-Rong GR

Synthetic and systems biotechnology 20221112 1

Betanin is a member of natural pigment betacyanins family and has extensive application in the food industry as an important natural red food colorant. Its relatively inefficient production in nature however hampers access to this phytochemicals through traditional crop-based manufacturing. Microbial bioproduction therefore represents an attractive alternative. Here, we present the construction of a <i>Saccharomyces cerevisiae</i> strain for betanin production. Through minimizing metabolic cross ...[more]

PMID: 36438069

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Project description:BackgroundWhole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels.ResultsHerein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions.ConclusionsAltogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

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.

Dataset Information

Improvement of betanin biosynthesis in Saccharomyces cerevisiae by metabolic engineering.

Publications

Improvement of betanin biosynthesis in <i>Saccharomyces cerevisiae</i> by metabolic engineering.

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