The efficiency of industrial fermentation process mainly depends on carbon yield, final titer and productivity. To improve the efficiency of l-lysine production from mixed sugar, we engineered carbohydrate metabolism systems to enhance the effective use of sugar in this study. A functional metabolic pathway of sucrose and fructose was engineered through introduction of fructokinase from Clostridium acetobutylicum. l-lysine production was further increased through replacement of phosphoenolpyruvate-dependent glucose and fructose uptake system (PTS Glc and PTS Fru ) by inositol permeases (IolT1 and IolT2) and ATP-dependent glucokinase (ATP-GlK). However, the shortage of intracellular ATP has a significantly negative impact on sugar consumption rate, cell growth and l-lysine production. To overcome this defect, the recombinant strain was modified to co-express bifunctional ADP-dependent glucokinase (ADP-GlK/PFK) and NADH dehydrogenase (NDH-2) as well as to inactivate SigmaH factor (SigH), thus reducing the consumption of ATP and increasing ATP regeneration. Combination of these genetic modifications resulted in an engineered C. glutamicum strain K-8 capable of producing 221.3 ± 17.6 g/L l-lysine with productivity of 5.53 g/L/h and carbon yield of 0.71 g/g glucose in fed-batch fermentation. As far as we know, this is the best efficiency of l-lysine production from mixed sugar. This is also the first report for improving the efficiency of l-lysine production by systematic modification of carbohydrate metabolism systems.

Background NAD(H/ + ) and NADP(H/ + ) are the most important redox cofactors in bacteria. However, the intracellular redox balance is in advantage of the cell growth and production of NAD(P)H-dependent products. Results In this paper, we rationally engineered glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and isocitrate dehydrogenase (IDH) to switch the nucleotide-cofactor specificity resulting in an increase in final titer [from 85.6 to 121.4 g L −1 ] and carbon yield [from 0.33 to 0.46 g (g glucose) −1 ] of l -lysine in strain RGI in fed-batch fermentation. To do this, we firstly analyzed the production performance of original strain JL-6, indicating that the imbalance of intracellular redox was the limiting factor for l -lysine production. Subsequently, we modified the native GAPDH and indicated that recombinant strain RG with nonnative NADP-GAPDH dramatically changed the intracellular levels of NADH and NADPH. However, l -lysine production did not significantly increase because cell growth was harmed at low NADH level. Lastly, the nonnative NAD-IDH was introduced in strain RG to increase the NADH availability and to equilibrate the intracellular redox. The resulted strain RGI showed the stable ratio of NADPH/NADH at about 1.00, which in turn improved cell growth (μ max. = 0.31 h −1 ) and l -lysine productivity ( q Lys, max. = 0.53 g g −1 h −1 ) as compared with strain RG (μ max. = 0.14 h −1 and q Lys, max. = 0.42 g g −1 h −1 ). Conclusions This is the first report of balancing the intracellular redox state by switching the nucleotide-cofactor specificity of GAPDH and IDH, thereby improving cell growth and l -lysine production. Electronic supplementary material The online version of this article (10.1186/s12934-019-1114-0) contains supplementary material, which is available to authorized users.

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Dehydrogenase pathway, one of diaminopimelate pathway, is important to the biosynthesis of L-lysine and peptidoglycan via one single reaction catalyzed by meso-diaminopimelate dehydrogenase (DapDH). In this study, the thermostable DapDH was introduced into diaminopimelate pathway that increased the final titer (from 71.8 to 119.5 g/L), carbon yield (from 35.3% to 49.1%) and productivity (from 1.80 to 2.99 g/(L∙h)) of L-lysine by LATR12-2∆rpiB::ddhSt in fed-batch fermentation. To do this, the kinetic properties and the effects of different DapDHs on L-lysine production were investigated, and the results indicated that overexpression of StDapDH in LATR12-2 was beneficial to construct an L-lysine producer with good productive performance because it exhibited the best of kinetic characteristics and optimal temperature as well as thermostability in reductive amination. Furthermore, ammonium availability was optimized, and found that 20 g/L of (NH4)2SO4 was the optimal ammonium concentration for improving the efficiency of L-lysine production by LATR12-2∆rpiB::ddhSt. Metabolomics analysis showed that introducing the StDapDH significantly enhanced carbon flux into pentose phosphate pathway and L-lysine biosynthetic pathway, thus increasing the levels of NADPH and precursors for L-lysine biosynthesis. This is the first report of a rational modification of diaminopimelate pathway that improves the efficiency of L-lysine production through overexpression of thermostable DapDH in E. coli.

This article focuses on engineering Corynebacterium glutamicum to produce L-lysine efficiently from starch using combined method of "classical breeding" and "genome breeding." Firstly, a thermo-tolerable L-lysine-producing C. glutamicum strain KT 45-6 was obtained after multi-round of acclimatization at high temperature. Then, amylolytic enzymes were introduced into strain KT 45-6 , and the resultant strains could use starch for cell growth and L-lysine production except the strain with expression of isoamylase. In addition, co-expression of amylolytic enzymes showed a good performance in starch degradation, cell growth and L-lysine production, especially co-expression of α-amylase (AA) and glucoamylase (GA). Moreover, L-lysine yield was increased by introducing AA-GA fusion protein (i.e., strain KT 45-6 S-5), and finally reached to 23.9 ± 2.3 g/L in CgXII IP M-medium. It is the first report of an engineered L-lysine-producing strain with maximum starch utilization that may be used as workhorse for producing amino acid using starch as the main feedstock. Key points• Thermo-tolerable C. glutamicum was obtained by temperature-induced adaptive evolution.• The fusion order between AA and GA affects the utilization efficiency of starch. • C. glutamicum with starch utilization was constructed by optimizing amylases expression.

BACKGROUND Starch can be used as a carbon source for producing menaquinone‐7 (MK‐7) by Bacillus sp., but the final titer and productivity are unsatisfactory because starch cannot be effectively hydrolyzed to fermentable sugars. This article focuses on developing thermo‐tolerable MK‐7 high‐producing strains by temperature‐induced mutagenesis‐based adaptive evolution. RESULTS Here, we isolated a thermo‐tolerable MK‐7 high‐producing strain B. amyloliquefaciens MK50‐36 after multi‐rounds of acclimation at high temperature. Strain MK50‐36 showed a large growth with soluble starch as carbon source at 50 °C. Compared with the parental strain B. amyloliquefaciens H.β.D.R.‐5 cultivated at 37 °C, MK‐7 yield of strain MK50‐36 increased about twofold at 50 °C when soluble starch was used as the sole carbon resource (17.5 ± 1.5 mg L–1 versus 37.1 ± 2.1 mg L–1). In addition, strain MK50‐36 showed an improvement in starch utilization rate as compared with strain H.β.D.R.‐5 because α‐amylase activity of strain MK50‐36 was the highest at 50 °C. Moreover, MK‐7 yield of strain MK50‐36 was further increased by feeding fresh broth to control the amount of fermentation liquid in fed‐batch fermentation because fermentation broth will be easy to volatilize and thus affect the production of MK‐7 at high temperature, which reached up to 52.7 ± 4.6 mg L–1. CONCLUSION These results indicated that soluble starch can act as an effective carbon source for producing MK‐7, and strain MK50‐36 has a superior capacity to produce MK‐7 from soluble starch. © 2020 Society of Chemical Industry