A bottom-up approach towards a bacterial consortium for the biotechnological conversion of chitin to L-lysine

Abstract

Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc₂, chitin deacetylase to convert GlcNAc₂ into GlcN₂ and acetate, and glucosaminidase to cleave GlcN₂ into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc₂ or GlcN₂ to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Key Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.

Keywords: Chitin; Corynebacterium glutamicum; Cross-feeding; Escherichia coli; Microbial consortia; N-acetylglucosamine.

Fig. 1

Design of the synthetic mutualistic consortium with E. coli and C. glutamicum for l-lysine production with chitin as sole source of carbon and energy. (1) The substrate converter EcLPPLYSA (E. coli W3110 ΔnagE ΔmanXYZ ΔchbBCA ΔlysA Δlpp::CM) expresses heterologous enzymes for the degradation of chitin to glucosamine (GlcN) and acetate. (2) EcLPPLYSA can only use acetate as growth substrate because of deletions in uptake systems for the other chitin degradation products. (3) CgLYS4 (C. glutamicum DM1729 Δpta-ackA Δcat ΔldhA ΔaceAB ΔnanR) can only use GlcN as growth substrate and for the production of l-lysine because of deletions in acetate metabolism. (4) The consortium is co-stabilized by the lysine auxotrophy of EcLPPLYSA. GlcNAc: N-acetylglucosamine, Fru-6-P: fructose-6-phosphate, GlcN-6-P: glucosamine-6-phosphate, LysA: diaminopimelate decarboxylase, ChbBCA: PTS-system chitobiose-specific, NagE: PTS-system N-acetylglucosamine-specific EIICBA component, ManXYZ: mannose-specific PTS-system

Fig. 2

Growth of the E. coli wild type strain EcWT (red circles) and its mutant strain EcLPPLYSA* (blue squares) on 20 mM (a) sodium acetate, (b) glucosamine, (c) N-acetylglucosamine, or (d) chitobiose. Error bars (mostly smaller than symbols) indicate standard deviation of three independent experiments (n = 3)

Fig. 3

Growth of C. glutamicum wild type strain DM1729 (red circles), and its mutant strain CgLYS4 (blue squares) on 20 mM (a) sodium acetate, (b) glucosamine, or (c) N-acetyl-glucosamine. Error bars indicate standard deviation of three independent experiments (n = 3)

Fig. 4

Growth of the E coli substrate converter strain EcLPP or EcLPPLYSA and the C. glutamicum producer strain CgLYS4 in single and co-cultures with a mixture of 40 mM glucosamine and 40 mM acetate as sole carbon and energy source. (a) CFUs of strain EcLPP (blue squares) and EcLPPLYSA (green triangles) in single culture (open symbols) and in co-culture with CgLYS4 (closed symbols). (b) CFUs of strain CgLYS4 in single culture (orange dots), in co-culture with ECLPP (blue dots) and in co-culture with EcLPPLYSA (green dots). Error bars indicate standard error of the mean of three independent experiments with triplicate determinations each (n = 3)

Fig. 5

HPTLC analysis of culture supernatants after 0–6 days. Culture supernatant of a culture of EcLPP* [empty] and of a culture of EcLPP* [TkCDA] supplied with 40 mM of GlcNAc, at time points t0d-t6d. Application volumes were 15 μl and 4 μl for samples and standards, respectively. Marker: standard GlcNAc-GlcNAc₆ or GlcN-GlcN₆ (each 2 mg ml⁻¹). Identification of GlcNAc and GlcNAc₂ was verified using UHPLC-ESI-MSⁿ (Supplemental Fig. S2)

Fig. 6

Growth of the E. coli substrate converter strain EcLPPLYSA* [TkCDA] expressing a functional chitin deacetylase or the control strain EcLPP* [empty] and of the C. glutamicum producer strain CgLYS4 in co-cultures with 40 mM GlcNAc as sole carbon and energy source. CFUs of strain (a) EcLPPLYSA* [TkCDA] (blue squares) or EcLPP* [empty] (red triangles) and (b) CgLYS4 in co-culture with EcLPPLYSA* [TkCDA] (blue dots) and in co-culture with EcLPP* [empty] (red dots). Error bars indicate standard error of the mean of three independent experiments with triplicate determinations each (n = 3)

Fig. 7

Growth of CgLYS4 with 40 mM GlcNAc as sole carbon and energy source with cell-free culture supernatant of EcLPP* [TkCDA] (green dots) or supernatant of an M9extra medium blank with 40 mM acetate (blue dots) and a control without addition of supernatant (red dots). Error bars indicate standard error of the mean of three independent experiments with triplicate determinations each (n = 3)

Fig. 8

Growth of E. coli strain EcLPPLYSA* [TkCDA] and C. glutamicum strain CgLYS4 in co-culture with 40 mM GlcNAc as sole carbon and energy source. CFU of strain (a) EcLPPLYSA* [TkCDA] and (b) CgLYS4. Red symbols represent CFU of co-culture with addition of IPTG, green symbols represent CFU without addition of IPTG and blue symbols with addition of culture supernatant of EcLPPLYSA* [TkCDA] cells and no IPTG. Error bars indicate standard error of the mean of three independent experiments with triplicate determinations each (n = 3). (c) Activity of TkCDA of supernatants taken from co-cultures with (green, open bars) and without IPTG (blue, filled bars) at t0d and t5d measured by determination of acetate

Fig. 9

Growth of E. coli strain EcCHB and C. glutamicum strain CgLYS4 in co-cultures with 0.5% (w/v) colloidal chitin as sole carbon and energy source. (a) CFUs of strain EcCHB with (filled bars) and without (open bars) addition of enzymes. (b) CFUs of strain CgLYS4 with (filled bars) and without (open bars) addition of enzymes. Addition of purified enzymes: 15 μg ml⁻¹ chitinase ChiB, 22.5 μg ml⁻¹ TK, and 7.5 μg ml⁻¹ TkCDA. Error bars indicate standard error of the mean (n = 2); n.s: not significant, *: statistically significant at P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 10

HPTLC analysis of supernatants from cultures of E. coli strains EcLPP* [ChiB] (a) and EcLPP* [TK] (b) as well as the empty vector control EcLPP* [empty]. (a) Glc: glucose standard (54 μg), marker: standard GlcNAc-GlcNAc₆ or GlcN-GlcN₄ (8 μg each), EcLPP* [empty]: culture supernatant of a culture of EcLPP* [empty] supplied with 0.1% (w/v) colloidal chitin and 20 mM Glc, at time points t0d-t6d. EcLPP* [ChiB]: culture supernatant of a culture of EcLPP* [ChiB] supplied with 0.1% (w/v) colloidal chitin and 20 mM Glc, at time points t0d-t6d. (b) marker: standard GlcNAc-GlcNAc₆ or GlcN-GlcN₆ (8 μg each), EcLPP* [empty]: culture supernatant of a culture of EcLPP* [empty] supplied with 12 mM GlcN₂ and 20 mM Glc, at time points t0-t6. EcLPP* [TK]: culture supernatant of a culture of EcLPP* [TK] supplied with 12 mM GlcN₂ and 20 mM Glc, at time points t0d-t6d. Application volume of all samples was 15 μl. Identification of Glc, GlcN, GlcN₂, and GlcNAc₂ was verified using UHPLC-ESI-MSⁿ (Supplemental Fig. S3)