Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

Background Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. Main text Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. Conclusions Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

A herbicide resistance risk assessment for weeds in maize in New Zealand

Despite an extensive history of research into herbicide resistance in New Zealand maize, some aspects remain understudied. Herbicide resistance was first detected in New Zealand in the 1980s in maize crops, with atrazine resistance in Chenopodium album L. and Persicaria maculosa Gray. Since then, Chenopodium album has also developed resistance to dicamba, and in the last five years Digitaria sanguinalis (L.) Scop. populations have been reported to be resistant to nicosulfuron. Here we estimate the risk of herbicide resistance arising in 39 common maize weeds. A list of weeds associated with maize was generated, omitting uncommon weeds and those that grow outside of the maize growing season. Weeds were ranked for their risk of evolving herbicide resistance with a scoring protocol that accounts for the specific herbicides used in New Zealand maize. Seven weed species were classified as having a high risk of developing herbicide resistance: Echinochloa crus-galli (L.) P.Beauv., Chenopodium album, Eleusine indica (L.) Gaertn., Xanthium strumarium L., Amaranthus powellii S.Watson, Solanum nigrum L. and Digitaria sanguinalis. Seventeen species were classed as moderate risk, and 15 were low risk. Herbicide classes associated with more resistant species were classed as high risk,these included acetohydroxy acid synthase inhibitors and photosystem-II inhibitors. Synthetic auxins had a moderate risk but only two herbicides in this class (dicamba and clopyralid) are registered for maize in New Zealand. Other herbicide mode-of-action groups used in maize were low risk. We recommend outreach to farmers regarding weed-control strategies that prevent high-risk species from developing resistance. High-risk herbicide groups should be monitored for losses of efficacy. Resistance surveys should focus on these species and herbicides.

Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

ABSTRACTWe previously demonstrated efficientl-valine production by metabolically engineeredCorynebacterium glutamicumunder oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis ofl-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase fromLysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase geneldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress thel-valine yield. Eliminating these by-products therefore was deemed key to improving thel-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase geneppc, succinate production was effectively suppressed, but both glucose consumption andl-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, includinggapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higherl-valine yield and productivity. Deleting the alanine transaminase geneavtAsuppressed alanine production. The resultant strain produced 1,280 mMl-valine at a yield of 88% mol mol of glucose−1after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

ABSTRACTProduction ofl-valine under oxygen deprivation conditions byCorynebacterium glutamicumlacking the lactate dehydrogenase geneldhAand overexpressing thel-valine biosynthesis genesilvBNCDEwas repressed. This was attributed to imbalanced cofactor production and consumption in the overalll-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole ofl-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement forl-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded byilvCand introduction ofLysinibacillus sphaericusleucine dehydrogenase in place of endogenous transaminase B, encoded byilvE. The intracellular NADH/NAD+ratio significantly decreased, and glucose consumption andl-valine production drastically improved. Moreover,l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor forl-valine production under oxygen deprivation conditions. Thel-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded byilvBN. The resultant strain produced 1,470 mMl-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and thel-valine productivity reached 1,940 mM after 48 h.