L-citrulline production by metabolically engineered Corynebacterium glutamicum from glucose and alternative carbon sources

L-citrulline plays an important role in human health and nutrition and is an intermediate of the L-arginine biosynthetic pathway. L-citrulline is a by-product of L-arginine production by Corynebacterium glutamicum. In this study, C. glutamicum was engineered for overproduction of L-citrulline as major product without L-arginine being produced as by-product. To this end, L-arginine biosynthesis was derepressed by deletion of the arginine repressor gene argR and conversion of L-citrulline towards L-arginine was avoided by deletion of the argininosuccinate synthetase gene argG. Moreover, to facilitate L-citrulline production the gene encoding a feedback resistant N-acetyl L-glutamate kinase argBfbr as well as the gene encoding L-ornithine carbamoylphosphate transferase argF were overexpressed. The resulting strain accumulated 44.1 ± 0.5 mM L-citrulline from glucose minimal medium with a yield of 0.38 ± 0.01 g⋅g−1 and a volumetric productivity of 0.32 ± 0.01 g⋅l−1⋅h−1. In addition, production of L-citrulline from the alternative carbon sources starch, xylose, and glucosamine could be demonstrated.


Introduction
L-citrulline is a natural non-proteinogenic amino acid whose name is derived from watermelon Citrullus lanatus (Wada 1930). In mammalians it serves as a precursor for L-arginine. In contrast to the proteinogenic L-arginine, which is not transferred to the blood stream, when ingested, L-citrulline can be converted to L-arginine, which is then released by the kidney into the blood stream. It is applied in several medical approaches e.g. as a pharmaconutrient (Rimando and Perkins-Veazie 2005;Curis et al. 2005).
Currently, biocatalytic and fermentative methods to produce L-citrulline using Pseudomonas putida (Kakimoto et al. 1971;Yamamoto et al. 1974) or Bacillus subtilis strains exist (Okumura et al. 1966). Additionally, extraction processes from watermelon have been established (Fish 2012). L-citrulline is an intermediate of L-arginine biosynthesis and accumulates as a by-product of engineered L-arginine producing Corynebacterium glutamicum strains (Ikeda et al. 2009;Schneider et al. 2011).
C. glutamicum is a workhorse for amino acid production and is employed for the annual production of several million tons of L-glutamate and L-lysine (Wendisch 2014). C. glutamicum has been engineered to produce a wide range of bioproducts, such as diamines, carotenoids, terpenes, proteins (Schneider and Wendisch 2010;Schneider et al. 2012;Heider et al. 2014a, b;Frohwitter et al. 2014;Kikuchi et al. 2009;Teramoto et al. 2011;An et al. 2013) and the L-glutamate family amino acids L-arginine, L-ornithine, and L-proline (Schneider et al. 2011;Ikeda et al. 2009;Georgi et al. 2005;Blombach et al. 2009;Jensen and Wendisch 2013). However, the production of L-citrulline as the only or major product has not been published yet.
Due to its natural ability to produce L-glutamate under several eliciting conditions, C. glutamicum is a suitable producer of L-glutamate-derived products (Sato et al. 2008;Radmacher et al. 2005;Kim et al. 2009Kim et al. , 2010Delaunay et al. 1999;Wendisch et al. 2014). L-ornithine is a non-proteinogenic glutamate-family amino acid and an intermediate of L-arginine biosynthesis ( Figure 1). An ornithine producer was obtained by deletion of argR, the gene encoding the genetic repressor of the arginine biosynthesis operon, and argF to prevent further processing of ornithine (Schneider et al. 2011). The production of L-proline from L-ornithine is possible by the heterologous overexpression of ocd from Pseudomonas putida, encoding ornithine cyclodeaminase (Jensen and Wendisch 2013). The diamine putrescine can be produced by overexpression of the Escherichia coli gene speC, which encodes ornithine decarboxylase (Schneider et al. 2012;Schneider and Wendisch 2010). As the arginine biosynthetic pathway is naturally regulated by feedback inhibition of N-acetylglutamate kinase (encoded by argB) by arginine, the use of feedback resistant enzyme variants in combination with deletion of argR has been described to overproduce L-arginine (Sakanyan et al. 1996;Ikeda et al. 2009;Schneider et al. 2011).
C. glutamicum can utilize a variety of carbon sources. In contrast to many other microorganisms used in biotechnology, simultaneous utilization of carbon sources e.g. present in mixtures such as lignocellulosic hydrolysates is a hall mark of C. glutamicum (Blombach and Seibold 2010;Meiswinkel et al. 2013a, b). The natural substrate spectrum of C. glutamicum includes monosaccharides, disaccharides, and organic acids as well as alcohols (Blombach and Seibold 2010;Arndt and Eikmanns 2008;Peters-Wendisch et al. 1998;Jolkver et al. 2009;Sasaki et al. 2011). To allow access to alternative carbon sources, C. glutamicum has also been engineered for utilization of glycerol, pentoses, and amino sugars as well as polysaccharides (Schneider et al. 2011;Rittmann et al. 2008;Seibold et al. 2006;Uhde et al. 2013;Gopinath et al. 2011;Matano et al. 2014).
One aim to reduce production cost is the use of complex sugar substrates for the production of biotechnological products. As an example of using a polymeric raw material without decomposition to its monomeric compounds e.g. by enzyme treatment, soluble starch could be used as a carbon source for the production of L-lysine and organic acids by engineered C. glutamicum   The cultivation was performed in CGXII minimal medium containing 20 g L-1 glucose, 1 mM IPTG, 750 μM L-arginine and 25 μg L-1 kanamycin. OD 600 was determined of CIT0(pVWEx1) (open squares), CIT0(pVWEx1-argF) (gray circles) and CIT0 (pVWEx1-argFB fbr ) (black diamonds). Values and error bars represent the mean and the standard error of triplicates. (Seibold et al. 2006;Tateno et al. 2007;Tsuge et al. 2013). However, due to the growing world population and a correlating higher demand for food, biotechnological processes based on non-food derived carbon sources are sought. Xylose is a pentose sugar compound present in the hemicellulosic fraction of agricultural wastes as for example rice straw. Glucosamine, on the other hand, is a constituent of chitin, the second most abundant biopolymer in Nature, which is accessible e.g. from shrimp shell waste accumulating in the food industry. C. glutamicum has been engineered to efficiently utilize both xylose and glucosamine as alternative carbon sources for growth and amino acid production (Gopinath et al. 2011;Meiswinkel et al. 2013a;Uhde et al. 2013;Matano et al. 2014).
In this study, the rational engineering of L-citrulline production by C. glutamicum is reported and the concept was extended to production of L-citrulline from the alternative carbon sources glucosamine, xylose, and starch.

Microorganisms and growth conditions
Microorganisms and plasmids used in this study are listed in Table 1. E. coli DH5α was used for gene cloning. C. glutamicum and E. coli strains were routinely grown in lysogeny broth (LB) (10 g L −1 tryptone, 5 g L −1 yeast extract, 10 g L −1 sodium chloride) in 500-mL baffled flasks on a rotary shaker (120 rpm) at 30°C or 37°C. For growth experiments, CGXII minimal medium (Eggeling and Reyes 2005) was used for C. glutamicum. Growth was followed by measuring the optical density at 600 nm using a V-1200 Spectrophotometer (VWR, Radnor, PA, USA). An OD 600 of 1 corresponds approximately to an estimated cell dry weight of 0.25 g/L.
When necessary, the growth medium was supplemented kanamycin (25 μg mL −1 ), spectinomycin (100 μg mL −1 ), tetracycline (10 μg mL −1 ), isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) and L-arginine (750 μM). The growth behavior and L-citrulline production of recombinant C. glutamicum strains were analyzed in 500 ml baffled flasks. Briefly, a 50 mL BHI (37 g L −1 ) seed culture was inoculated from an agar plate and grown overnight. The cells were harvested by centrifugation (4,000 × g, 10 min) and washed twice with CGXII minimal medium lacking the carbon source. Subsequently, 50 mL CGXII medium, containing a given concentration of carbon source and necessary supplements, was inoculated to an optical density of 1.0. Detailed information on the carbon source concentrations employed are given in the Results chapter.

Molecular genetic techniques
Standard methods such as restriction digestions, and ligation were carried out as described elsewhere (Sambrook and Russell 2012). Digested DNA was purified by using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Table 1 Strains and plasmids used in this study  (Seibold et al. 2006) E. coli cells were transformed by heat shock (Sambrook and Russell 2012) and C. glutamicum cells were transformed by electroporation (Eggeling and Reyes 2005). Isolation of genomic DNA was performed as previously described (Jensen and Wendisch 2013). Chromosomal changes in C. glutamicum were performed as described elsewhere (Eggeling and Reyes 2005).

Construction of strains and plasmids
The deletion of ΔargFR in MB001 was performed by using pK19mobsacBΔargFR. Afterwards argG was deleted by using pK19mobsacBΔargG to obtain CIT0. pK19mobsacBΔargG contains the up-and downstream regions of argG in the ΔargFR strain. The plasmid was constructed by amplifying the upstream region with argG_up_f (CTTgaattcAGAAGCTGCGCCGCATG) and argG_up_r (agagacgacctaagccagtctAACGATGCGGTTAGTCATGAGG) and the downstream region with argG_down_f (agactggct taggtcgtctctGCTAACAAGCGCGATCGC) and argG_down_r (CCTctgcagAACGACCAGCGCGCAGA). The two fragments were combined by crossover PCR using argG_up_f and argG_down_r and finally cloned into pK19mobsacB with PstI and EcoRI. pVWEx1-argF was constructed by amplifying argF with primers argF_f (CTTgtcgacAAGGAGATATAGATATGAC TTCACAACCACAGGTTCG) and argF_r (CCTggatccTT ACCTCGGCTGGTTGGC). The PCR product was treated with SalI and BamHI and ligated with similarly treated pVWEx1. pVWEx1-argFG was constructed by amplifying argG with primers argG_f (GGGgtcgacGAAAGG AGGCCCTTCAGATGACTAACCGCATCGTTCTTG) and argG_r (GGGgtcgacTTAGTTGTTGCCAGCTTCG CGA). The PCR product was treated with SalI and ligated with similarly treated pVWEx1-argF.
The plasmid vector pEKEx-argB fbr (argB A49VM54V (Schneider et al. 2011)) was digested with BamHI and KpnI and the DNA fragment with a size of 0.9 kb harboring the argB fbr gene was cloned into the BamHI/KpnI digested vector pVWEx1-argF.

Determination of amino acid and carbohydrate concentrations
For the quantification of extracellular amino acids and carbohydrates, a high-performance liquid chromatography system was used (1200 series, Agilent Technologies Deutschland GmbH, Böblingen, Germany). Samples were withdrawn from the cultures, centrifuged (13,000 × g, 10 min), and the supernatant used for analysis.

Results
Engineering a prophage-free C. glutamicum strain for L-citrulline production C. glutamicum has recently been cured of prophage sequences to yield MB001 (Baumgart et al. 2013). This strain was used as the parental strain because it can be transformed easily and plasmid-based gene overexpression is more efficient (Baumgart et al. 2013). As C. glutamicum ATCC 13032, this strain does not accumulate L-citrulline, an intermediate of L-arginine biosynthesis (Figure 1). The deletion of three genes of the L-arginine operon (L-ornithine carbamoyltransferase (EC 2.1.3.3) argF, argininosuccinate synthetase (EC 6.3.4.5) argG, and L-arginine biosynthesis operon repressor gene argR) in C. glutamicum MB001 yielded the L-arginine auxotrophic strain CIT0 (Table 1). When supplemented with 0.75 mM L-arginine, C. glutamicum CIT0 accumulated 25.2 ± 2.6 mM L-ornithine from 2% glucose ( Table 2). The deletion of argF and argG could be complemented by plasmid-borne expression of these genes since the complemented strain CIT0(pVWEx1-argFG) grew without L-arginine supplement while the empty vector carrying control CIT0(pVWEx1) did not (data not shown). Comparable growth rates and biomass concentrations were observed.

Production of L-citrulline from alternative carbon sources
Due to the high demand of biotechnological processes of using complex sugar substrates derived from raw materials and industrial wastes, the L-citrulline producer strain CIT1 was enabled to utilize the alternative carbon sources starch (as an example of a high molecular weight carbohydrate), xylose, and glucosamine (as an example of a carbohydrates, derived from forestry and food industrial wastes).
To enable C. glutamicum CIT1 to consume starch, the gene amyA from Streptomyces griseus was overexpressed. The combined overexpression of xylA from Xanthomonas campestris and endogenous xylB allowed the utilization of xylose by C. glutamicum CIT1. The endogenous nagB was overpressed ectopically to facilitate the consumption of glucosamine. The resulting strains were tested for growth and L-citrulline production.
As the determination of the starch concentration by HPLC was not possible, residual starch content was assayed by the use of Lugols solution. However, as it is known that overexpression of amyA in C. glutamicum results in high molecular mass degradation products of starch, which remain in the medium and are not detectable by Lugols solution (Seibold et al. 2006), the L-citrulline concentration was measured until no change in OD 600 , starch content and L-citrulline concentration was observed. The starch utilizing strain CIT1(pAmy) was able to produce 11.9 ± 0.5 mM L-citrulline which corresponds to a yield of 0.167 g/g.