A Novel Strategy for D-Psicose and Lipase Co-Production Using a Co

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A Novel Strategy for D-Psicose and Lipase Co-Production Using a Co Zhang et al. Bioresour. Bioprocess. (2021) 8:77 https://doi.org/10.1186/s40643-021-00429-8 RESEARCH Open Access A novel strategy for D-psicose and lipase co-production using a co-culture system of engineered Bacillus subtilis and Escherichia coli and bioprocess analysis using metabolomics Jun Zhang1,2,4 , Wen Luo1, Zhiyuan Wang1, Xiaoyan Chen1,4, Pengmei Lv1* and Jingliang Xu1,3* Abstract To develop an economically feasible fermentation process, this study designed a novel bioprocess based on the co- culture of engineered Bacillus subtilis and Escherichia coli for the co-production of extracellular D-psicose and intracel- lular lipase. After optimizing the co-culture bioprocess, 11.70 g/L of D-psicose along with 16.03 U/mg of lipase was obtained; the glucose and fructose were completely utilized. Hence, the conversion rate of D-psicose reached 69.54%. Compared with mono-culture, lipase activity increased by 58.24%, and D-psicose production increased by 7.08%. In addition, the co-culture bioprocess was explored through metabolomics analysis, which included 168 carboxylic acids and derivatives, 70 organooxygen compounds, 34 diazines, 32 pyridines and derivatives, 30 benzene and substituted derivatives, and other compounds. It also could be found that the relative abundance of diferential metabolites in the co-culture system was signifcantly higher than that in the mono-culture system. Pathway analysis revealed that, tryp- tophan metabolism and β-alanine metabolism had the highest correlation and played an important role in the co- culture system; among them, tryptophan metabolism regulates protein synthesis and β-alanine metabolism, which is related to the formation of metabolic by-products. These results confrm that the co-cultivation of B. subtilis and E. coli can provide a novel idea for D-psicose and lipase biorefnery, and are benefcial for the discovery of valuable second- ary metabolites such as turanose and morusin. Highlights Co-cultivation was frstly carried out for the co-production of D-psicose and lipase. The carbon source glucose and fructose were fully utilized during co-culture. The yield of D- psicose was 11.70 g/L, and the conversion rate reached 69.54%. Self-induced expression of lipase was realized with enzyme activity of 16.03 U/mg. Metabolomics analyses were used to explore the co-culture efects of mixed strains. Keywords: D-Psicose, Lipase, Escherichia coli, Bacillus subtilis, Co-culture, Metabolomics *Correspondence: [email protected]; [email protected] Introduction 1 Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou Institute of Energy Conversion, D-Psicose is a rare sugar and a new type of functional Chinese Academy of Sciences, CAS Key Laboratory of Renewable Energy, sweetener with excellent physiological efects such as Guangzhou 510640, China Full list of author information is available at the end of the article anti-hyperglycemic and anti-hyperlipidemic efects © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Zhang et al. Bioresour. Bioprocess. (2021) 8:77 Page 2 of 18 (Chen et al. 2019; Zhang et al. 2016). Terefore, it is mixed microorganisms with complementary advan- widely used in food, medicine, and other felds. Presently, tages (Bertrand et al. 2014; Rosero-Chasoy et al. 2021). enzymatic conversion is the most important method for Furthermore, it has a strong ability to tolerate harsh the synthesis of D-psicose, and under the catalytic action environments, such as infection, substrate, and prod- of ketose 3-epimerases, D-fructose can be isomerized uct inhibition; hence, better fermentation results can into D-psicose, which is the current hot spot of D-psicose be obtained. Currently, there are two main forms of co- synthesis (Jiang et al. 2020). However, the use of fer- culture systems (Wein et al. 2019): (a) the co-cultivation mentation methods for the cultivation of engineered of unknown microbial fora isolated from nature and (b) Bacillus subtilis, which harbors the D-tagatose 3-epime- the co-cultivation of artifcially designed microbial fora. rase (DTEase) gene, has the potential to directly produce Compared with the former, the latter system is relatively D-psicose as reported in a previous study (Zhang et al. fragile; therefore, there should be a careful selection of 2020a). Te conversion rate of the substrate can reach the combined strains. To ensure the efcient and stable 56.26%, which is a great advantage in realizing the cou- growth of the co-culture system, the population ratio pling of cell growth and D-psicose production, thereby in a reasonable range and external conditions at certain reducing the biosynthesis costs. stages, which are favored by priority populations, has Lipase is a hydrolase which acts on the ester bond with been maintained through optimized medium compo- catalytic diversity, and it is also one of the most widely nents and strengthened process regulation (Klitgord and used biocatalysts in industrial production (Melani et al. Segre 2010). B. subtilis and E. coli are Gram-positive and 2020). Geobacillus thermocatenulatus lipase 2 (GTL2) is Gram-negative bacteria, respectively. Tey have a well- a typical thermophilic enzyme with high catalytic activity mapped genetic background and are commonly used as and good thermal stability and has several applications model organisms. Under optimized co-culture condi- in pharmaceutical and organic synthesis, chiral com- tions, they can produce efective synergistic efects and pound resolution, and bioenergy; thus, it is a promising increase the yield of target products (Faust and Raes biocatalyst with great application potential (Godoy et al. 2012). Recently, indole (an aromatic heterocyclic organic 2019; Kajiwara et al. 2020). In addition, Escherichia coli compound) was produced by co-cultivation of E. coli and has a short culture cycle with high target protein levels, B. subtilis with signifcantly improved yield (Singh et al. which makes it a good host for expressing GTL2. Under 2020). the induction of expensive IPTG, GTL2 achieved solu- Te construction of an artifcial co-culture system is ble expression in E. coli, and the enzyme activity reached relatively easy and simple, but metabolic regulation is rel- 39.50 U/mg in a previous study (Zhang et al. 2020b). atively complicated. Tus, it is necessary to use metabo- However, to enhance the fermentation economy, it is lomics analysis to explore the symbiotic efects of mixed desirable to induce GTL2 expression using α-lactose. strains. In general, metabolomics is defned as the study Presently, single-bacterial fermentation of lipase may of the overall metabolite spectrum in a system (cell, tis- cause excessive production costs, whereas mixed-bac- sue, or organism) under given conditions. It is a collection terial fermentation of expensive D-psicose can reduce of small molecular metabolites with a molecular weight the fermentation costs. Tis innovation in traditional of less than 1000 in biological samples (Patti et al. 2012). fermentation not only reduces the overall cost, but also A large amount of qualitative and quantitative metabolite improves the quality of fermentation. In addition, co- data can be obtained and classifed according to specifc culture bioprocess can also efectively utilize glucose and metabolic pathways to construct metabolic networks fructose, which is of relevance for complex substrates, and infer their physiological functions to be able to more such as waste fruit and vegetable hydrolysate (Jiang et al. comprehensively analyze the dynamic metabolic reac- 2017), to achieve the goal of carbon neutrality, and the tions of organisms in response to environmental changes. full consumption of the carbon source is conducive to the Microbial metabolites, as well as the identifcation of subsequent separation of D-psicose. Furthermore, in pre- metabolic variations in diferent environments, can easily vious studies, the extracellular production of D-psicose be screened through efective analytical methods, such in recombinant B. subtilis (Zhang et al. 2020a) and the as liquid chromatography–mass spectrometry (LC–MS) soluble expression of GTL2 in engineered E. coli (Zhang based on the UHPLC–QE-MS nontargeted metabo- et al. 2020b) have been achieved. Hence, it is better to lomics approach (Cheng et al. 2020; Xia et al. 2020). explore the possibility of co-production of D-psicose and However, there is limited information about the difer- lipase using co-culture to develop a more economical fer- ential metabolites and key metabolic pathways in the mentation process. co-culture process of B. subtilis and E. coli in the exist- Based on this principle of symbiosis (Wein et al. 2019), ing literature. Hence, the screening of major diferential the co-culture of microorganisms is used to ferment metabolites and metabolic pathways using metabolomics Zhang et al. Bioresour. Bioprocess. (2021) 8:77 Page 3 of 18 analysis can better manipulate the co-cultivation process protein solution obtained was subjected to protein level of B. subtilis and E. coli, which is benefcial to the stable analysis using SDS-PAGE. operation of the co-culture system. In this study, a co-culture system of recombinant B. Lipase activity analysis subtilis and E.
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