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Application of a Pyruvate-Producing Escherichia Coli Strain Lafcpcpt-Accbc-Acee: a Case Study for D-Lactate Production

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Application of a Pyruvate-Producing Escherichia Coli Strain Lafcpcpt-Accbc-Acee: a Case Study for D-Lactate Production fermentation Article Application of a Pyruvate-Producing Escherichia coli Strain LAFCPCPt-accBC-aceE: A Case Study for d-Lactate Production Keisuke Wada 1,2 , Tatsuya Fujii 1,*, Hiroyuki Inoue 1, Hironaga Akita 1, Tomotake Morita 2 and Akinori Matsushika 1,3 1 Research Institute for Sustainable Chemistry (RISC), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-hiroshima, Hiroshima 739-0046, Japan; [email protected] (K.W.); [email protected] (H.I.); [email protected] (H.A.); [email protected] (A.M.) 2 Research Institute for Sustainable Chemistry (RISC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan; [email protected] 3 Graduate School of Integrated Science for Life, Hiroshima University, 1-3-1 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8530, Japan * Correspondence: [email protected]; Tel.: +81-82-493-6843 Received: 11 June 2020; Accepted: 16 July 2020; Published: 17 July 2020 Abstract: Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d-lactate dehydrogenase-encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 1.4 g L 1, which was compatible with the pyruvate production by the PYR strain ± · − (26.1 0.9 g L 1). These results suggest that we have succeeded in the effective conversion of pyruvate ± · − to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production. Keywords: Escherichia coli; pyruvate; fermentation; d-Lactate; NADH/NAD+ ratio 1. Introduction Pyruvate is one of the key metabolites in the central metabolism of all known organisms, and has been studied extensively for industrial use. The typical example of the use of pyruvate itself is the compound’s use in human health, for instance, in protecting cells against oxidative stress [1]. In addition, pyruvate is used as a precursor to various other chemicals, such as amino acids [2], alcohols [3], plastic monomers [4], and pharmaceuticals [5–7] (Figure1). Three methods of pyruvate production are known: chemical synthesis, fermentation, and enzymatic production [2]. Among the three, the fermentation method is known to have the lowest costs of raw materials, and commercial pyruvate production by fermentation has been developed in various organisms [2,8–10]. These facts indicate that it is possible to construct various chemical-producing strains based on a pyruvate-producing strains. Fermentation 2020, 6, 70; doi:10.3390/fermentation6030070 www.mdpi.com/journal/fermentation Fermentation 2020, 6, x FOR PEER REVIEW 2 of 10 indicate that it is possible to construct various chemical-producing strains based on a pyruvate- Fermentation 2020, 6, 70 2 of 10 producing strains. Figure 1. Derivatives of pyruvate. IPP and DMAPP indicate isopentenyl diphosphate and dimethylallyl Figure 1. Derivatives of pyruvate. IPP and DMAPP indicate isopentenyl diphosphate and pyrophosphate, respectively. dimethylallyl pyrophosphate, respectively. To enhance the productivity by strains of various chemicals, including derivatives of pyruvate, a rangeTo ofenhance metabolic the productivity modifications by of strains the producer of variou microbess chemicals, have beenincluding attempted. derivatives The regulationof pyruvate, of intracellulara range of metabolic redox balance modifications is one of theof the key producer strategies, microbes given that have cofactors been NAD(P)attempted.+ and The NAD(P)H regulation play of + importantintracellular roles redox in microbial balance metabolismis one of the [ 11key]. Forstrategies, example, given the inactivationthat cofactors of aldehydeNAD(P) dehydrogenaseand NAD(P)H toplay prevent important competition roles forin NADHmicrobial with metabolism ethanol-forming [11]. reactionsFor example, improved the 1,3-propanediolinactivation of production aldehyde withdehydrogenaseKlebsiella pneumoniae to preventby 2-foldcompetition [12]. In for another NADH example, with ethanol-forming the aeration control reactions to prevent improved competition 1,3- forpropanediol NADH with production the respiratory with Klebsiella chain improved pneumoniaeL-valine by 2-fold production [12]. In with anotherCorynebacterium example, the glutamicum aeration bycontrol 20-fold to prevent [13]. Therefore, competition the regulation for NADH of with intracellular the respiratory redox balance chain improved is important L-valine for the production microbial productionwith Corynebacterium of chemicals, glutamicum although by the 20-fold specific [13]. modifications Therefore, the needed regulation depend of onintracellular the production redox conditions,balance is important including for the the nature microbial of the hostproduction strains andof chemicals, the targeted although products. the specific modifications neededIn adepend previous on the report, production we described conditions, the including construction the nature of a pyruvate-producing of the host strains andE. the coli targetedstrain, LAFCPCPt-accBC-aceEproducts. (designated here as PYR) [14]. The genes accBC and aceE, encoding acetyl-CoA carboxylaseIn a previous and pyruvate report, dehydrogenase, we described respectively,the construction are known of a topyruvate-producing be essential for cell E. growth coli strain, when utilizingLAFCPCPt-accBC-aceE glucose as the sole (designated carbon source here [15 as]. PYR) Hence, [14]. in the The PYR genes strain, accBC the expressionand aceE, ofencodingaccBC and acetyl-aceE wasCoA controlled carboxylase by and a doxycycline-inducible pyruvate dehydrogenase, system respecti [14,16vely,]. The are silencing known ofto beaccBC essentialand aceE for cellexpression growth usingwhen thisutilizing regulatory glucose system as the was sole an carbon effective source mechanism [15]. Hence, for inducing in the PYR pyruvate strain, production the expression [14,15 of]. Furthermore,accBC and aceE in was the controlled PYR strain, by severala doxycycline-induci genes were disruptedble system to [14,16]. enhance The pyruvate silencing productivity,of accBC and withaceE targetsexpression including: using this (i) biosynthetic regulatory system genes involved was an ineffective the production mechanism of ethanol, for inducing lactate, pyruvate acetate, andproduction formate; [14,15]. and (ii) Furthermore,cra, a gene thatin the encodes PYR strain a global, several transcription genes were regulator disrupted (Figure to enhance2). In pyruvate simple batchproductivity, culture, thewith PYR targets strain including: showed high (i) biosynthet pyruvate productivityic genes involved (26.1 in0.9 the g L production1), demonstrating of ethanol, 56% ± · − elactate,fficiency acetate, per consumed and formate; glucose and when (ii) cra glucose, a gene was that used encodes as the a global sole carbon transcription source [ 14regulator]. These (Figure results −1 indicated2). In simple that PYRbatch is aculture, promising the foundationPYR strain strain showed for thehigh production pyruvate of productivity pyruvate derivatives. (26.1 ± 0.9 As g·L a first), exampledemonstrating of the application56% efficiency of the per PYR consumed strain, a d -lactate-producingglucose when glucose strain was (designated used as herethe assole old-LAC) carbon wassource constructed [14]. These by results introducing indicated a Lactobacillus that PYR is bulgaricum a promisinggene foundation encoding strain NAD+ for-dependent the productiond-lactate of dehydrogenasepyruvate derivatives. (DLDH) As into a first the example PYR background of the application [17]. The d of-lactate the PYR production strain, a pathwayD-lactate-producing was shown strain (designated here as old-LAC) was constructed by introducing a Lactobacillus bulgaricum gene1 in Figure2. However, the d-lactate production of the old-LAC strain was low level (1.1 0.0 g L− ) encoding NAD+-dependent D-lactate dehydrogenase (DLDH) into the1 PYR background [17].± The· D- compared with pyruvate production by the PYR strain (26.1 0.9 g L− ), implying that the expression lactate production pathway was shown in Figure 2. However,± the· D-lactate production of the old- of DLHD in the old-LAC strain was insufficient. These results indicate that there is room for further LAC strain was low level (1.1 ± 0.0 g·L−1) compared with pyruvate production by the PYR strain (26.1 improvement of the d-lactate production by the PYR-derived strain. ± 0.9 g·L−1), implying that the expression of DLHD in the old-LAC strain was insufficient. These results indicate that there is room for further improvement of the D-lactate production by the PYR-derived strain. Fermentation 2020, 6, 70 3 of 10 Fermentation 2020, 6, x FOR PEER REVIEW 3 of 10 Figure 2. Schematic metabolic pathway of the d-lactate-producing
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